Islet cell manufacturing compositions and methods of use

ABSTRACT

Disclosed herein are compositions and methods useful for manufacturing SC-β cell, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

CROSS-REFERENCE

This application is a continuation of international application No.PCT/US2018/061364, filed on Nov. 15, 2018, which claims the benefit ofU.S. Provisional Application No. 62/586,808, filed on Nov. 15, 2017, andU.S. Provisional Application No. 62/669,170, filed on May 9, 2018, eachof which is incorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Diabetes is a major healthcare problem globally. Approximately, morethan 400 million people suffer from diabetes and tis complicationsworldwide as reported by International Diabetes Federation in 2015.Among them, more than 50 million people require insulin injections.Death or dysfunction of pancreatic β cells in pancreatic islets whichleads to abnormal insulin secretion can cause diabetes. The generationof stem cell derived β-cells can provide a potentially useful steptoward the generation of islets and pancreatic organs, which canpotentially provide therapeutic treatment of diabetes. One of therapidly growing diseases that may be treatable by stem cell derivedtissues is diabetes. Type I diabetes can result from autoimmunedestruction of β-cells in the pancreatic islet. Type II diabetes canresult from peripheral tissue insulin resistance and β-cell dysfunction.Diabetic patients, particularly those suffering from type I diabetes,can potentially be cured through transplantation of new β-cells.Patients transplanted with cadaveric human islets can be made insulinindependent for 5 years or longer via this strategy, but this approachis limited because of the scarcity and quality of donor islets. Thegeneration of an unlimited supply of human β-cells from stem cells canextend this therapy to millions of new patients and can be an importanttest case for translating stem cell biology into the clinic.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in some aspects, is a method for generating a cellcluster that comprises Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells, the method comprising: contacting a population ofPdx1-negative, NKX6.1-negative primitive gut tube cells with acomposition comprising a bone morphogenetic protein (BMP) signalingpathway inhibitor and a growth factor from transformation growth factorβ (TGF-β) superfamily, thereby generating a cell cluster that comprisesPdx1-positive, NKX6.1-positive pancreatic progenitor cells, wherein thecluster comprises at most about 30% CHGA-positive cells and at mostabout 30% CDX2-positive cells as measured by flow cytometry.

In some embodiments, the method further comprises differentiating thePDX1-positive/NKX6.1-negative pancreatic progenitor cells intopancreatic β cells.

In some embodiments, the method further comprises differentiating thePDX1-positive/NKX6.1-negative pancreatic progenitor cells intoPDX1-positive/NKX6.1-positive pancreatic progenitor cells.

In some embodiments, the method further comprises differentiating theNKX6.1-positive pancreatic progenitor cells into insulin-positiveendocrine cells.

In some embodiments, the method further comprises differentiating theinsulin-positive endocrine cells into pancreatic β cells.

Disclosed herein, in some aspects, is a method comprising: (a)contacting a population of cells comprising a Pdx1-positive,NKX6.1-positive primitive gut tube cell with a composition comprising aBMP signaling pathway inhibitor and a growth factor from TGF-βsuperfamily, thereby generating a cell cluster comprising aPdx1-positive, NKX6.1-positive pancreatic progenitor cell; and (b)differentiating the cell cluster comprising thePDX1-positive/NKX6.1-positive pancreatic progenitor cell into a cellcluster comprising non-native pancreatic β cells, wherein the cellcluster comprising non-native pancreatic β cells has aglucose-stimulated insulin secretion (GSIS) stimulation index higherthan a comparable cell cluster generated without the contacting with theBMP signaling pathway inhibitor and the growth factor from TGF-βsuperfamily.

In some embodiments, the GSIS stimulation index of the cell cluster isat least about 1.2 fold, at least about 1.5 fold, at least about 1.8fold, at least about 2 fold, at least about 2.2 fold, at least about 2.4fold, at least about 2.8 fold, or at least about 3 fold higher than thatof the comparable cell cluster. In some embodiments, the GSISstimulation index of the cell cluster is at least about 3 fold higherthan that of the comparable population. In some embodiments, the GSISstimulation index is calculated as a ratio of insulin secretion inresponse to a first glucose concentration to insulin secretion inresponse to a second glucose concentration. In some embodiments, thefirst glucose concentration is about 10 to about 50 mM, and the secondglucose concentration is about 1 mM to 5 mM. In some embodiments, thefirst glucose concentration is about 20 mM, and the second glucoseconcentration is about 2.8 mM. In some embodiments, the cell clustercomprises a higher percentage of the non-native pancreatic β cell thanthe comparable cell cluster as measured by flow cytometry. In someembodiments, the cell cluster comprises a percentage of the non-nativepancreatic β cell at least about 1.1 fold, at least about 1.2 fold, atleast about 1.3 fold, at least about 1.4 fold, or at least about 1.5fold higher than the comparable cell cluster as measured by flowcytometry. In some embodiments, the cell cluster comprises a percentageof the non-native pancreatic β cells about 1.5 fold higher than thecomparable cell cluster as measured by flow cytometry.

Disclosed herein, in some aspects, is a method comprising: (a)contacting a population of cells comprising a Pdx1-positive,NKX6.1-positive primitive gut tube cell with a composition comprising aBMP signaling pathway inhibitor and a growth factor from TGF-βsuperfamily, thereby generating a cell cluster comprising aPdx1-positive, NKX6.1-positive pancreatic progenitor cell; and (b)differentiating the cell cluster comprising thePDX1-positive/NKX6.1-positive pancreatic progenitor cell into a cellcluster comprising non-native pancreatic β cells, wherein the cellcluster comprising non-native pancreatic β cells comprises a higherpercentage of the non-native pancreatic β cell, as measured by flowcytometry, as compared to a comparable cell cluster generated withoutthe contacting with the BMP signaling pathway inhibitor and the growthfactor from TGF-β superfamily.

In some embodiments, the cell cluster comprising the non-nativepancreatic β cells comprises a percentage of the non-native pancreatic βcells at least about 1.1 fold, at least about 1.2 fold, at least about1.3 fold, at least about 1.4 fold, or at least about 1.5 fold higherthan the comparable cell cluster as measured by flow cytometry. In someembodiments, the cell cluster comprising the non-native pancreatic βcells comprises a percentage of the non-native pancreatic β cells about1.5 fold higher than the comparable cell cluster as measured by flowcytometry. In some embodiments, the cell cluster comprising thenon-native pancreatic β cells exhibits a higher insulin secretion inresponse to a glucose challenge as compared to the comparable cellcluster. In some embodiments, the cell cluster comprising the non-nativepancreatic β cells exhibits at least about 1.2, 1.5, 2, 2.5, 3, 3.5, 4,4.5, or 5 fold higher an insulin secretion in response to a glucosechallenge as compared to the comparable cell cluster. In someembodiments, the cell cluster comprising the non-native pancreatic βcells exhibits a higher GSIS stimulation index as compared to thecomparable cell cluster. In some embodiments, the GSIS stimulation indexof the cell cluster comprising the non-native pancreatic β cells is atleast about 1.2 fold, at least about 1.5 fold, at least about 1.8 fold,at least about 2 fold, at least about 2.2 fold, at least about 2.4 fold,at least about 2.8 fold, or at least about 3 fold higher than that ofthe comparable cell cluster. In some embodiments, the GSIS stimulationindex of the cell cluster comprising the non-native pancreatic β cellsis at least about 3 fold higher than that of the comparable cellcluster. In some embodiments, the GSIS stimulation index is calculatedas a ratio of insulin secretion in response to a first glucoseconcentration to insulin secretion in response to a second glucoseconcentration. In some embodiments, the first glucose concentration isabout 10 to about 50 mM, and the second glucose concentration is about 1mM to 5 mM. In some embodiments, the first glucose concentration isabout 20 mM, and the second glucose concentration is about 2.8 mM. Insome embodiments, the non-native pancreatic β cells exhibit an in vitroglucose-stimulated insulin secretion response when exposed to a glucosechallenge. In some embodiments, the non-native pancreatic β cellsexhibit an insulin secretion in response to a first concentration of K⁺.In some embodiments, the cell cluster comprising the non-nativepancreatic β cells exhibits a higher insulin secretion as compared tothe comparable cell cluster in response to a first concentration of K⁺.In some embodiments, the cell cluster comprising the non-nativepancreatic β cells exhibits at least about 1.2 fold, at least about 1.5fold, at least about 1.8 fold, at least about 2 fold, at least about 2.2fold, at least about 2.4 fold, at least about 2.8 fold, at least about 3fold, at least about 3.2 fold, at least about 3.4 fold, at least about3.6 fold, at least about 3.8 fold, at least about 4 fold higher aninsulin secretion as compared to the comparable cell cluster in responseto a first concentration of K⁺. In some embodiments, the inhibitor ofthe BMP signaling pathway comprises DMH-1, a derivative, analogue, orvariant thereof. In some embodiments, the composition comprises about0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, about 0.1 μM toabout 1 μM, or about 0.15 μM to about 0.5 μM DMH-1. In some embodiments,the composition comprises about 0.25 μM DMH-1. In some embodiments, thegrowth factor from TGF-β superfamily comprises Activin A. In someembodiments, the composition comprises about 0.5 ng/mL to about 200ng/mL, about 1 ng/mL to about 100 ng/mL, about 2 ng/mL to about 50ng/mL, or about 5 ng/mL to about 30 ng/mL Activin A. In someembodiments, the composition comprises at least about 5 ng/mL or atleast about 10 ng/mL Activin A. In some embodiments, the compositioncomprises about 20 ng/mL Activin A. In some embodiments, the compositionfurther comprises a differentiation factor selected from the groupconsisting of: a growth factor from FGF family, a SHH pathway inhibitor,a RA signaling pathway activator, a protein kinase C activator, and aROCK inhibitor.

Disclosed herein, in some aspects, is a method comprisingdifferentiating a population of cells comprising Pdx1-negative,NKX6.1-negative primitive gut tube cell in a culture medium comprisingabout 0.01% (w/v) to about 0.5% (w/v) human serum albumin (HSA), therebygenerating a cell cluster comprising Pdx1-positive, NKX6.1-negativepancreatic progenitor cells.

In some embodiments, at least about 60%, at least about 70%, or at leastabout 85% of cells in the cell cluster comprising the Pdx1-positive,NKX6.1-negative pancreatic progenitor cells are Pdx1-positive asmeasured by flow cytometry. In some embodiments, at least about 85% ofcells in the cell cluster comprising the Pdx1-positive, NKX6.1-negativepancreatic progenitor cells are Pdx1-positive as measured by flowcytometry. In some embodiments, at most about 40%, at most about 30%, atmost about 20%, or at most about 15% of cells in the cell clustercomprising the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells are CDX2-positive cells as measured by flow cytometry. In someembodiments, at most about 15% of cells in the cell cluster comprisingthe Pdx1-positive, NKX6.1-negative pancreatic progenitor cells areCDX2-positive cells as measured by flow cytometry. In some embodiments,the culture medium further comprises a differentiation factor selectedfrom the group consisting of: a BMP signaling pathway inhibitor, agrowth factor from TGF-β superfamily, a growth factor from FGF family, aSHH pathway inhibitor, a RA signaling pathway activator, a proteinkinase C activator, and a ROCK inhibitor. In some embodiments, theculture medium further comprises a BMP signaling pathway inhibitor and agrowth factor from TGF-β superfamily.

Disclosed herein, in some aspects, is a method comprising: (a) culturinga population of cells comprising a primitive gut tube cell in a culturemedium comprising a bone morphogenetic protein (BMP) signaling pathwayinhibitor, a growth factor from transformation growth factor β (TGF-β)superfamily, and human serum albumin (HSA), thereby generating a cellcluster comprising a Pdx1-positive, NKX6.1-positive pancreaticprogenitor cell; and (b) differentiating the cell cluster comprising thePDX1-positive, NKX6.1-positive pancreatic progenitor cell into a cellcluster comprising a non-native pancreatic β cell.

Disclosed herein, in some aspects, is a cell cluster comprising at leastabout 50% Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, atmost about 30% chromogranin A (CHGA)-positive cells, and at most about30% CDX2-positive cells as measured by flow cytometry.

In some embodiments, the cell cluster comprises at most about 25% theCDX2-positive, NKX6.1-positive cells as measured by flow cytometry. Insome embodiments, the cell cluster comprises at most about 20% theCDX2-positive, NKX6.1-positive cells as measured by flow cytometry. Insome embodiments, the cell cluster comprises at most about 25% theCHGA-positive cells as measured by flow cytometry. In some embodiments,the cell cluster comprises at most about 10% the CHGA-positive cells asmeasured by flow cytometry. In some embodiments, the cell clustercomprises at most about 5% the CHGA-positive cells as measured by flowcytometry. In some embodiments, the cell cluster comprises at leastabout 60% the Pdx1-positive, NKX6.1-positive pancreatic progenitor cellsas measured by flow cytometry. In some embodiments, the cell clustercomprises at least about 65% the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells as measured by flow cytometry.

In some embodiments, further differentiation of the cell cluster resultsin a first cell cluster comprising non-native pancreatic β cells thathas a higher glucose-stimulated insulin secretion (GSIS) stimulationindex than a second cell cluster comprising the non-native pancreatic βcells differentiated from a comparable cell cluster comprising at leastabout 50% the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells, and more than 30% the chromogranin A (CHGA)-positive cells ormore than 30% the CDX2-positive cells as measured by flow cytometry.

Disclosed herein, in some aspects, is a composition comprising the cellcluster disclosed herein and a bone morphogenetic protein (BMP)signaling pathway inhibitor and a growth factor from transformationgrowth factor β (TGF-β) superfamily. In some embodiments, the inhibitorof BMP signaling pathway comprises DMH-1 or derivative thereof. In someembodiments, the composition comprises about 0.01 μM to about 10 μM,about 0.05 μM to about 5 μM, about 0.1 μM to about 1 μM, or about 0.15μM to about 0.5 μM DMH-1. In some embodiments, the composition comprisesabout 0.25 μM DMH-1. In some embodiments, the growth factor from TGF-βsuperfamily comprises Activin A. In some embodiments, the compositioncomprises about 0.5 ng/mL to about 200 ng/mL, about 1 ng/mL to about 100ng/mL, about 2 ng/mL to about 50 ng/mL, or about 5 ng/mL to about 30ng/mL Activin A. In some embodiments, the composition comprises at leastabout 5 ng/mL or at least about 10 ng/mL Activin A. In some embodiments,the composition comprises about 20 ng/mL Activin A. In some embodiments,the composition further comprises a differentiation factor selected fromthe group consisting of: a growth factor from fibroblast growth factor(FGF) family, a Sonic Hedgehog (SHH) pathway inhibitor, a retinoic acid(RA) signaling pathway activator, a protein kinase C activator, and aRho-associated protein kinase (ROCK) inhibitor. In some embodiments, thecell cluster is in a culture medium. In some embodiments, thecomposition further comprises about 0.01% (w/v) to about 0.5% (w/v)human serum albumin (HSA). In some embodiments, the composition furthercomprises about 0.05% (w/v) HSA.

Disclosed herein, in some aspects, is a cell cluster comprising at leastabout 60% Pdx1-positive, NKX6.1-negative pancreatic progenitor cells andat most about 40% CDX2-positive cells as measured by flow cytometry.

In some embodiments, the cell cluster comprises at least about 70% thePdx1-positive, NKX6.1-negative pancreatic progenitor cells as measuredby flow cytometry. In some embodiments, the cell cluster comprises atleast about 85% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells as measured by flow cytometry. In some embodiments, the cellcluster comprises at most about 30% the CDX2-positive cells as measuredby flow cytometry. In some embodiments, the cell cluster comprises atmost about 20% the CDX2-positive cells as measured by flow cytometry. Insome embodiments, the cell cluster comprises at most about 15% theCDX2-positive cells as measured by flow cytometry.

In some embodiments, further differentiation of the cell cluster resultsin a first cell cluster comprising non-native pancreatic β cells thathas a higher glucose-stimulated insulin secretion (GSIS) stimulationindex than a second cell cluster comprising the non-native pancreatic βcells differentiated from a comparable cell cluster comprising at leastabout 60% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells, and more than 40% the CDX2-positive cells as measured by flowcytometry.

Disclosed herein, in some aspects, is a composition comprising the cellcluster disclosed herein in a culture medium comprising human serumalbumin. In some embodiments, the culture medium comprises about 0.01%(w/v) to about 0.5% (w/v) HSA. In some embodiments, the culture mediumfurther comprises a differentiation factor selected from the groupconsisting of: a BMP signaling pathway inhibitor, a growth factor fromTGF-β superfamily, a growth factor from FGF family, a SHH pathwayinhibitor, a RA signaling pathway activator, a protein kinase Cactivator, and a ROCK inhibitor.

Disclosed herein, in some aspects, is a cell cluster comprisingnon-native pancreatic β cells, wherein the cell cluster is obtained fromdifferentiation of primitive gut tube cells by contacting the primitivegut tube cells with a bone morphogenetic protein (BMP) signaling pathwayinhibitor and a growth factor from transformation growth factor 13(TGF-β) superfamily, and wherein the cell cluster has a higher number ofthe non-native pancreatic β cells per cubic micrometer as compared to acomparable cell cluster obtained from differentiation of primitive guttube cells without the contacting.

In some embodiments, the cell cluster has at least about 1.1, 1.2, 1.3,1.4, 1.5, or 1.6 fold an higher number of the non-native pancreatic βcells per cubic micrometer as compared to the comparable cell cluster.

Disclosed herein, in some aspects, is a cell cluster comprisingnon-native pancreatic β cells, wherein the cell cluster is obtained fromdifferentiation of primitive gut tube cells by contacting the primitivegut tube cells with a bone morphogenetic protein (BMP) signaling pathwayinhibitor and a growth factor from transformation growth factor 13(TGF-β) superfamily, and wherein the cell cluster exhibits higherinsulin secretion in response to glucose challenge as compared to acomparable cell cluster obtained from differentiation of primitive guttube cells without the contacting.

In some embodiments, the cell cluster exhibits at least about 1.2, 1.5,2, 2.5, 3, 3.5, 4, 4.5, or 5 fold higher an insulin secretion ascompared to the comparable cell cluster. In some embodiments, the cellcluster exhibits a higher GSIS stimulation index as compared to thecomparable cell cluster. In some embodiments, the GSIS stimulation indexof the cell cluster is at least about 1.2 fold, at least about 1.5 fold,at least about 1.8 fold, at least about 2 fold, at least about 2.2 fold,at least about 2.4 fold, at least about 2.8 fold, or at least about 3fold higher than that of the comparable cell cluster. In someembodiments, the GSIS stimulation index of the cell cluster comprisingthe non-native pancreatic β cells is at least about 3 fold higher thanthat of the comparable cell cluster. In some embodiments, the GSISstimulation index is calculated as a ratio of insulin secretion inresponse to a first glucose concentration to insulin secretion inresponse to a second glucose concentration. In some embodiments, thefirst glucose concentration is about 10 to about 50 mM, and the secondglucose concentration is about 1 mM to 5 mM. In some embodiments, thefirst glucose concentration is about 20 mM, and the second glucoseconcentration is about 2.8 mM. In some embodiments, the non-nativepancreatic β cells exhibit an in vitro glucose-stimulated insulinsecretion response when exposed to a glucose challenge. In someembodiments, the non-native pancreatic β cells exhibit an insulinsecretion in response to a first concentration of K⁺. In someembodiments, the cell cluster exhibits a higher insulin secretion ascompared to the comparable cell cluster in response to a firstconcentration of K⁺. In some embodiments, the cell cluster exhibits atleast about 1.2 fold, at least about 1.5 fold, at least about 1.8 fold,at least about 2 fold, at least about 2.2 fold, at least about 2.4 fold,at least about 2.8 fold, at least about 3 fold, at least about 3.2 fold,at least about 3.4 fold, at least about 3.6 fold, at least about 3.8fold, at least about 4 fold higher an insulin secretion as compared tothe comparable cell cluster in response to a first concentration of K⁺.

Disclosed herein, in some aspects, is a cell cluster comprisingnon-native pancreatic β cells produced according to the method disclosedherein.

Disclosed herein, in some aspects, is a pharmaceutical compositioncomprising a cell cluster comprising non-native pancreatic β cellsproduced according to the method disclosed herein.

Disclosed herein, in some aspects, is a pharmaceutical compositioncomprising the cell cluster disclosed herein.

Disclosed herein, in some aspects, is a device comprising the cellcluster disclosed herein or a cell cluster comprising non-nativepancreatic β cells produced according to the method disclosed herein,wherein the device is configured to produce and release insulin whenimplanted into a subject.

In some embodiments, the device further comprises a semipermeablemembrane, wherein the semipermeable membrane is configured to retaincells in the device and permit passage of insulin secreted by the cells.In some embodiments, the cells are encapsulated by the semipermeablemembrane. In some embodiments, the semipermeable membrane is made ofpolysaccharide or polycation. In some embodiments, the semipermeablemembrane is made of a material selected from the group consisting of:poly(lactide) (PLA), poly(glycolic acid) (PGA),poly(lactide-co-glycolide) (PLGA), other polyhydroxyacids,poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyphosphazene, polyamino acids, polyortho esters, polyacetals,polycyanoacrylates, polytetrafluoroethylene (PTFE), biodegradablepolyurethanes, albumin, collagen, fibrin, polyamino acids, prolamines,alginate, agarose, agarose with gelatin, dextran, polyacrylates,ethylene-vinyl acetate polymers and other acyl-substituted celluloseacetates and derivatives thereof, polyurethanes, polystyrenes, polyvinylchloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonatedpolyolefins, polyethylene oxide, and any combinations thereof. In someembodiments, the semipermeable membrane comprises alginate. In someembodiments, the cell cluster is encapsulated in a microcapsule thatcomprises an alginate core surrounded by the semipermeable membrane.

Disclosed herein, in some aspects, is a method of treating a subject,comprising administering the subject with non-native pancreatic β cellsproduced according to the method disclosed herein, the cell clusterdisclosed herein, the pharmaceutical composition disclosed herein, orthe device disclosed herein.

In some embodiments, the subject has, or has an increased risk ofdeveloping a metabolic disorder. In some embodiments, the subject hasdiabetes selected from the group consisting of: Type I diabetes, Type IIdiabetes, and Type 1.5 diabetes.

Provided herein, in some embodiments, is a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl ascompared to the amount of insulin secreted upon induction with glucose.In some embodiments, the population of glucose-responsive insulinsecreting cells secrete at least 1.5 times, 2 times, 2.5 times, 3 timeshigher amount of insulin upon induction with KCl as compared to theamount of insulin secreted upon induction with glucose. In someembodiments, the population of glucose-responsive insulin secretingcells is contacted with an amount of a signaling factor.

In some embodiments, the signaling factor is provided in an amountsufficient to result in an increase in insulin production as compared toa corresponding composition not contacted with the signaling factor. Insome embodiments, the increase is a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6 or 7 fold increase.

Also provided herein, in some embodiments, is a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl and/orglucose, in the presence of a signaling factor as compared to comparablecells in the absence of the signaling factor. In some embodiments, thecells secrete higher amount of insulin in the presence of high glucose,but not in the presence of low glucose.

Also provided herein, in some embodiments, is a population ofdifferentiated pancreatic progenitor cells, wherein the populationcomprises at least 60% pancreatic β cells as determined by flowcytometry. In some embodiments, the population comprises at least 65%,70%, 75%, 80%, 85%, or 90% pancreatic β cells.

In some embodiments, the population comprises a higher percentage ofpancreatic β cells upon being contacted with a predetermined basalmedium component as compared to a comparable population not contactedwith the basal medium component.

Also provided herein, in some embodiments, is a method comprisingimplanting in a subject a device comprising insulin producing cells,wherein the device releases insulin in an amount sufficient for areduction of blood glucose levels in the subject. In some embodiments,the insulin producing cells are glucose responsive insulin producingcells. In some embodiments, the reduction of blood glucose levels in thesubject results in an amount of glucose which is lower than the diabetesthreshold. In some embodiments, the subject is a mammalian subject. Insome embodiments, the mammalian subject is human. In some embodiments,the amount of glucose is reduced to lower than the diabetes threshold in1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the implanting.

Also provided herein, in some embodiments, is a method ofdifferentiating a population of progenitor cells into a population ofpancreatic β cells in vitro comprising culturing the population ofprogenitor cells in suspension in a culture medium comprising a basalmedium component wherein a percentage of the population of pancreatic βcells after differentiation is at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, or at least 99%. In some embodiments, thepopulation of progenitor cells is a population of embryonic stem cells.In some embodiments, the population of progenitor cells comprises asubpopulation of Oct4 expressing cells. In some embodiments, apercentage of the subpopulation of Oct4 expressing cells is at least90%. In some embodiments, the population of pancreatic β cells is apopulation of stem cell-derived β cells. In some embodiments, theculture medium comprises 0.1 L, 0.5 L, or 3 L of medium. In someembodiments, the population of pancreatic β cells after differentiationhas a stimulation index of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 or greater. In someembodiments, the stimulation index is determined in a conditioncomprising a signaling factor.

Provided herein, in some embodiments, is a composition of isolatedpancreatic β cells produced according to the disclosed methods. Providedherein is a pharmaceutical composition of isolated pancreatic β cellsproduced according to the disclosed methods. Provided herein is a methodof treating a subject, comprising administering the subject withisolated pancreatic β cells produced according to the disclosed methods.

Also disclosed herein, in some embodiments, is a composition comprisinga population of cells or cell cluster that comprises at least about 20%,30%, 40%, or 50% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, thepopulation of cells or cell cluster comprises at least about 40%, 50%,60%, 70%, 80%, or 85% NKX6.1⁺ cells. In some embodiments, the populationof cells or cell cluster comprises at least about 30%, 40%, 50%, or 55%C-peptide⁺ cells. In some embodiments, the population of cells or cellcluster comprises at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, or95% CHGA⁺ cells.

In some aspects, the disclosure relates to compositions and methods toscale up islet cell production. In some embodiments, the culture of stemcell can be scaled to up to 0.1 L, up to 0.2 L, up to 0.5 L, up to 1 L,up to 1.5 L, up to 2 L, up to 2.5 L, up to 3 L, up to 3.5 L, up to 4 L,up to 4.5 L, or up to 5 L. In some embodiments, the culture of stem cellcan be scaled to up to 6 L, 7 L, 8 L, 9 L, or 10 L.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 depicts three exemplary candidate cell lines for islet cellproduction.

FIG. 2A depicts morphology of an exemplary 2D culture of embryonic stemcells (ESCs).

FIG. 2B depicts percentage of ESCs expressing cell surface marker Sox17or Oct4 of an exemplary 2D culture.

FIGS. 3A and 3B depict examples flow cytometry data for on-target andoff-target progenitor populations.

FIGS. 4A and 4B depict a comparison of exemplary stem cell-derived β(SC-β) cells produced using 3 different protocols.

FIG. 5 depicts images of exemplary SC-β cells and natural pancreaticislet cells.

FIG. 6A depicts effects of exemplary differentiation factor (LDN) onStage 4 cells differentiated using an exemplary method disclosed herein.FIG. 6B depicts effects of exemplary differentiation factors (DMH-1 andActivin A) on Stage 4 cells differentiated using an exemplary methoddisclosed herein. FIG. 6C depicts an example result of glucosestimulated insulin secretion (GSIS) assay using produced SC-β cells atincreasing concentration of exemplary factors (DMH-1 and Activin A).

FIG. 7 depicts a comparison of insulin secretion of natural islet cellsand exemplary SC-β cells at low or high glucose stimulation.

FIG. 8 depicts process development goals for SC-β cell product.

FIG. 9A depicts a cell image of an exemplary 0.1 L culture of SC-βcells.

FIG. 9B depicts a cell image of an exemplary 0.5 L culture of SC-βcells.

FIG. 9C depicts a cell image of an exemplary 3.0 L culture of SC-βcells.

FIG. 9D depicts fold expansion of exemplary 0.1 L, 0.5 L, and 3 Lcultures of SC-β cells.

FIG. 9E depicts percentage of Oct4 expressing cells in the example 0.1L, 0.5 L, and 3 L cultures of SC-β cells.

FIG. 10 depicts a comparison of insulin secretion of cryopreserved SC-βcells and fresh SC-β cells.

FIG. 11 depicts an image of transplanted SC-islet graft at 6 months.

FIG. 12 depicts an example result of blood glucose level before or afterSC-β cell implant, and before or after SC-β cell explant in animalmodels.

FIG. 13 depicts a comparison of exemplary differentiated cellpopulations.

FIG. 14 depicts images of exemplary SC-islet cells before encapsulationand 3 months post-implant in a mouse.

FIG. 15 is a schematic illustrating one example of the effects ofexemplary BMP signaling pathway inhibitors LDN193189 (“LDN” in thefigure) and DMH-1.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of thepresent disclosure in detail. It is to be understood that thisdisclosure is not limited to the particular embodiments described hereinand as such can vary. Those of skill in the art will recognize thatthere are numerous variations and modifications of this disclosure,which are encompassed within its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described inthe context of a single embodiment, the features can also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure can be described herein in the context of separateembodiments for clarity, the present disclosure can also be implementedin a single embodiment.

Definitions

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It must be noted that, as used in thespecification, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

Use of the term “including” as well as other forms, such as “include,”“includes,” and “included,” is not limiting.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the present disclosure, and vice versa.Furthermore, compositions of the present disclosure can be used toachieve methods of the present disclosure.

The term “about” in relation to a reference numerical value and itsgrammatical equivalents as used herein can include the numerical valueitself and a range of values plus or minus 10% from that numericalvalue.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value. In anotherexample, the amount “about 10” includes 10 and any amounts from 9 to 11.In yet another example, the term “about” in relation to a referencenumerical value can also include a range of values plus or minus 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively,particularly with respect to biological systems or processes, the term“about” can mean within an order of magnitude, preferably within 5-fold,and more preferably within 2-fold, of a value. Where particular valuesare described in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

The term “diabetes” and its grammatical equivalents as used herein canrefer to is a disease characterized by high blood sugar levels over aprolonged period. For example, the term “diabetes” and its grammaticalequivalents as used herein can refer to all or any type of diabetes,including, but not limited to, type 1, type 2, cystic fibrosis-related,surgical, gestational diabetes, and mitochondrial diabetes. In somecases, diabetes can be a form of hereditary diabetes.

The term “endocrine cell(s),” if not particularly specified, can referto hormone-producing cells present in the pancreas of an organism, suchas “islet,” “islet cells,” “islet equivalent,” “islet-like cells,”“pancreatic islets” and their grammatical equivalents. In an embodiment,the endocrine cells can be differentiated from pancreatic progenitorcells or precursors. Islet cells can comprise different types of cells,including, but not limited to, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells, and/or pancreatic E cells. Isletcells can also refer to a group of cells, cell clusters, or the like.

The terms “progenitor” and “precursor” cell are used interchangeablyherein and can refer to cells that have a cellular phenotype that ismore primitive (e.g., is at an earlier step along a developmentalpathway or progression than is a fully differentiated cell) relative toa cell which it can give rise to by differentiation. Often, progenitorcells can also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

A “precursor thereof” as the term related to an insulin-positiveendocrine cell can refer to any cell that is capable of differentiatinginto an insulin-positive endocrine cell, including for example, apluripotent stem cell, a definitive endoderm cell, a primitive gut tubecell, a pancreatic progenitor cell, or endocrine progenitor cell, whencultured under conditions suitable for differentiating the precursorcell into the insulin-positive endocrine cell.

The terms “stem cell-derived β cell,” “SC-β cell,” “functional β cell,”“functional pancreatic β cell,” “mature SC-β cell,” and theirgrammatical equivalents can refer to cells (e.g., non-native pancreaticβ cells) that display at least one marker indicative of a pancreatic βcell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous mature β cell. In some embodiments, the terms “SC-β cell” and“non-native β cell” as used herein are interchangeable. In someembodiments, the “SC-β cell” comprises a mature pancreatic cell. It isto be understood that the SC-β cells need not be derived (e.g.,directly) from stem cells, as the methods of the disclosure are capableof deriving SC-β cells from any insulin-positive endocrine cell orprecursor thereof using any cell as a starting point (e.g., one can useembryonic stem cells, induced-pluripotent stem cells, progenitor cells,partially reprogrammed somatic cells (e.g., a somatic cell which hasbeen partially reprogrammed to an intermediate state between an inducedpluripotent stem cell and the somatic cell from which it was derived),multipotent cells, totipotent cells, a transdifferentiated version ofany of the foregoing cells, etc, as the invention is not intended to belimited in this manner). In some embodiments, the SC-β cells exhibit aresponse to multiple glucose challenges (e.g., at least one, at leasttwo, or at least three or more sequential glucose challenges). In someembodiments, the response resembles the response of endogenous islets(e.g., human islets) to multiple glucose challenges. In someembodiments, the morphology of the SC-β cell resembles the morphology ofan endogenous β cell. In some embodiments, the SC-β cell exhibits an invitro GSIS response that resembles the GSIS response of an endogenous βcell. In some embodiments, the SC-β cell exhibits an in vivo GSISresponse that resembles the GSIS response of an endogenous β cell. Insome embodiments, the SC-β cell exhibits both an in vitro and in vivoGSIS response that resembles the GSIS response of an endogenous β cell.The GSIS response of the SC-β cell can be observed within two weeks oftransplantation of the SC-β cell into a host (e.g., a human or animal).In some embodiments, the SC-β cells package insulin into secretorygranules. In some embodiments, the SC-β cells exhibit encapsulatedcrystalline insulin granules. In some embodiments, the SC-β cellsexhibit a stimulation index of greater than 1. In some embodiments, theSC-β cells exhibit a stimulation index of greater than 1.1. In someembodiments, the SC-β cells exhibit a stimulation index of greater than2. In some embodiments, the SC-β cells exhibit cytokine-inducedapoptosis in response to cytokines. In some embodiments, insulinsecretion from the SC-β cells is enhanced in response to knownantidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-βcells are monohormonal. In some embodiments, the SC-β cells do notabnormally co-express other hormones, such as glucagon, somatostatin orpancreatic polypeptide. In some embodiments, the SC-β cells exhibit alow rate of replication. In some embodiments, the SC-β cells increaseintracellular Ca2+ in response to glucose.

As used herein, the term “insulin producing cell” and its grammaticalequivalent can refer to a cell differentiated from a pancreaticprogenitor, or precursor thereof, which secretes insulin. Aninsulin-producing cell can include pancreatic β cell as that term isdescribed herein, as well as pancreatic β-like cells (e.g.,insulin-positive endocrine cells) that synthesize (e.g., transcribe theinsulin gene, translate the proinsulin mRNA, and modify the proinsulinmRNA into the insulin protein), express (e.g., manifest the phenotypictrait carried by the insulin gene), or secrete (release insulin into theextracellular space) insulin in a constitutive or inducible manner. Apopulation of insulin producing cells, e.g., produced by differentiatinginsulin-positive, endocrine cells or a precursor thereof into SC-β cellsaccording to the methods of the present disclosure can be pancreatic βcell or (β-like cells (e.g., cells that have at least one, or at leasttwo least two) characteristic of an endogenous β cell and exhibit aglucose stimulated insulin secretion (GSIS) response that resembles anendogenous adult β cell. The population of insulin-producing cells,e.g., produced by the methods as disclosed herein can comprise maturepancreatic β cell or SC-β cells, and can also containnon-insulin-producing cells (e.g. cells of cell like phenotype with theexception they do not produce or secrete insulin).

The terms “insulin-positive β-like cell,” “insulin-positive endocrinecell,” and their grammatical equivalents can refer to cells (e.g.,pancreatic endocrine cells) that displays at least one marker indicativeof a pancreatic β cell and also expresses insulin but lack a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous β cell.

The term “β cell marker” refers to, without limitation, proteins,peptides, nucleic acids, polymorphism of proteins and nucleic acids,splice variants, fragments of proteins or nucleic acids, elements, andother analyte which are specifically expressed or present in pancreaticβ cells. Exemplary β cell markers include, but are not limited to,pancreatic and duodenal homeobox 1 (Pdx1) polypeptide, insulin,c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, GLUT2, PC2,ZnT-8, Isl1, Pax6, Pax4, NeuroD, 1 Inflb, Hnf-6, Hnf-3beta, and MafA,and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001).

The term “pancreatic endocrine marker” can refer to without limitation,proteins, peptides, nucleic acids, polymorphism of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analyte which are specifically expressed or presentin pancreatic endocrine cells. Exemplary pancreatic endocrine cellmarkers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

The term “pancreatic progenitor,” “pancreatic endocrine progenitor,”“pancreatic precursor,” “pancreatic endocrine precursor” and theirgrammatical equivalents are used interchangeably herein and can refer toa stem cell which is capable of becoming a pancreatic hormone expressingcell capable of forming pancreatic endocrine cells, pancreatic exocrinecells or pancreatic duct cells. These cells are committed todifferentiating towards at least one type of pancreatic cell, e.g. βcells that produce insulin; a cells that produce glucagon; δ cells (or Dcells) that produce somatostatin; and/or F cells that produce pancreaticpolypeptide. Such cells can express at least one of the followingmarkers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “Pdx1-positive pancreatic progenitor” as used herein can referto a cell which is a pancreatic endoderm (PE) cell which has thecapacity to differentiate into SC-β cells, such as pancreatic β cells. APdx1-positive pancreatic progenitor expresses the marker Pdx1. Othermarkers include, but are not limited to Cdcp1, or Ptf1a, or HNF6 orNRx2.2. The expression of Pdx1 may be assessed by any method known bythe skilled person such as immunochemistry using an anti-Pdx1 antibodyor quantitative RT-PCR. In some cases, a Pdx1-positive pancreaticprogenitor cell lacks expression of NKX6.1. In some cases, aPdx1-positive pancreatic progenitor cell can also be referred to asPdx1-positive, NKX6.1-negative pancreatic progenitor cell due to itslack of expression of NKX6.1.

The term “Pdx1-positive, NKX6.1-positive pancreatic progenitor” as usedherein can refer to a cell which is a pancreatic endoderm (PE) cellwhich has the capacity to differentiate into insulin-producing cells,such as pancreatic β cells. A Pdx1-positive, NKX6.1-positive pancreaticprogenitor expresses the markers Pdx1 and NKX6.1. Other markers include,but are not limited to Cdcp1, or Ptf1a, or HNF6 or NRx2.2. Theexpression of NKX6.1 may be assessed by any method known by the skilledperson such as immunochemistry using an anti-NKX6.1 antibody orquantitative RT-PCR. In some cases, NKX6.1 protein or gene can also bereferred to as “NKX6-1” protein or gene.

The term “Ngn3-positive endocrine progenitor” as used herein can referto precursors of pancreatic endocrine cells expressing the transcriptionfactor Neurogenin-3 (Ngn3). Progenitor cells are more differentiatedthan multipotent stem cells and can differentiate into only few celltypes. In particular, Ngn3-positive endocrine progenitor cells have theability to differentiate into the five pancreatic endocrine cell types(α, β, δ, ε and PP). The expression of Ngn3 may be assessed by anymethod known by the skilled person such as immunochemistry using ananti-Ngn3 antibody or quantitative RT-PCR.

The terms “NeuroD” and “NeuroD I” are used interchangeably and identifya protein expressed in pancreatic endocrine progenitor cells and thegene encoding it.

The term “differentiated cell” or its grammatical equivalents is meantany primary cell that is not, in its native form, pluripotent as thatterm is defined herein. Stated another way, the term “differentiatedcell” can refer to a cell of a more specialized cell type derived from acell of a less specialized cell type (e.g., a stem cell such as aninduced pluripotent stem cell) in a cellular differentiation process.Without wishing to be limited to theory, a pluripotent stem cell in thecourse of normal ontogeny can differentiate first to an endoderm cellthat is capable of forming pancreas cells and other endoderm cell types.Further differentiation of an endoderm cell leads to the pancreaticpathway, where ^(˜)98% of the cells become exocrine, ductular, or matrixcells, and ˜2% become endocrine cells. Early endocrine cells are isletprogenitors, which can then differentiate further into insulin-producingcells (e.g. functional endocrine cells) which secrete insulin, glucagon,somatostatin, or pancreatic polypeptide. Endoderm cells can also bedifferentiate into other cells of endodermal origin, e.g. lung, liver,intestine, thymus etc.

As used herein, the term “somatic cell” can refer to any cells formingthe body of an organism, as opposed to germline cells. In mammals,germline cells (also known as “gametes”) are the spermatozoa and ovawhich fuse during fertilization to produce a cell called a zygote, fromwhich the entire mammalian embryo develops. Every other cell type in themammalian body—apart from the sperm and ova, the cells from which theyare made (gametocytes) and undifferentiated stem cells—is a somaticcell: internal organs, skin, bones, blood, and connective tissue are allmade up of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell,” by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell,” by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. Unless otherwiseindicated the methods for converting at least one insulin-positiveendocrine cell or precursor thereof to an insulin-producing, glucoseresponsive cell can be performed both in vivo and in vitro (where invivo is practiced when at least one insulin-positive endocrine cell orprecursor thereof are present within a subject, and where in vitro ispracticed using an isolated at least one insulin-positive endocrine cellor precursor thereof maintained in culture).

As used herein, the term “adult cell” can refer to a cell foundthroughout the body after embryonic development.

The term “endoderm cell” as used herein can refer to a cell which isfrom one of the three primary germ cell layers in the very early embryo(the other two germ cell layers are the mesoderm and ectoderm). Theendoderm is the innermost of the three layers. An endoderm celldifferentiates to give rise first to the embryonic gut and then to thelinings of the respiratory and digestive tracts (e.g. the intestine),the liver and the pancreas.

The term “a cell of endoderm origin” as used herein can refer to anycell which has developed or differentiated from an endoderm cell. Forexample, a cell of endoderm origin includes cells of the liver, lung,pancreas, thymus, intestine, stomach and thyroid. Without wishing to bebound by theory, liver and pancreas progenitors (also referred to aspancreatic progenitors) are develop from endoderm cells in the embryonicforegut. Shortly after their specification, liver and pancreasprogenitors rapidly acquire markedly different cellular functions andregenerative capacities. These changes are elicited by inductive signalsand genetic regulatory factors that are highly conserved amongvertebrates. Interest in the development and regeneration of the organshas been fueled by the intense need for hepatocytes and pancreatic βcells in the therapeutic treatment of liver failure and type I diabetes.Studies in diverse model organisms and humans have revealedevolutionarily conserved inductive signals and transcription factornetworks that elicit the differentiation of liver and pancreatic cellsand provide guidance for how to promote hepatocyte and β celldifferentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein can refer to a celldifferentiated from an endoderm cell and which can be differentiatedinto a SC-β cell (e.g., a pancreatic β cell). A definitive endoderm cellexpresses the marker Sox17. Other markers characteristic of definitiveendoderm cells include, but are not limited to MIXL2, GATA4, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99,CMKOR1 and CRIP1.

In particular, definitive endoderm cells herein express Sox17 and insome embodiments Sox17 and HNF3B, and do not express significant levelsof GATA4, SPARC, APF or DAB. Definitive endoderm cells are not positivefor the marker Pdx1 (e.g. they are Pdx1-negative). Definitive endodermcells have the capacity to differentiate into cells including those ofthe liver, lung, pancreas, thymus, intestine, stomach and thyroid. Theexpression of Sox17 and other markers of definitive endoderm may beassessed by any method known by the skilled person such asimmunochemistry, e.g., using an anti-Sox17 antibody, or quantitativeRT-PCR.

The term “pancreatic endoderm” can refer to a cell of endoderm originwhich is capable of differentiating into multiple pancreatic lineages,including pancreatic β cells, but no longer has the capacity todifferentiate into non-pancreatic lineages.

The term “primitive gut tube cell” or “gut tube cell” as used herein canrefer to a cell differentiated from an endoderm cell and which can bedifferentiated into a SC-β cell (e.g., a pancreatic β cell). A primitivegut tube cell expresses at least one of the following markers: HNP1-β,HNF3-β or HNF4-α. Primitive gut tube cells have the capacity todifferentiate into cells including those of the lung, liver, pancreas,stomach, and intestine. The expression of HNF1-β and other markers ofprimitive gut tube may be assessed by any method known by the skilledperson such as immunochemistry, e.g., using an anti-HNF1-β antibody.

The term “stem cell” as used herein, can refer to an undifferentiatedcell which is capable of proliferation and giving rise to moreprogenitor cells having the ability to generate a large number of mothercells that can in turn give rise to differentiated, or differentiabledaughter cells. The daughter cells themselves can be induced toproliferate and produce progeny that subsequently differentiate into oneor more mature cell types, while also retaining one or more cells withparental developmental potential. The term “stem cell” can refer to asubset of progenitors that have the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone embodiment, the term stem cell refers generally to a naturallyoccurring mother cell whose descendants (progeny) specialize, often indifferent directions, by differentiation, e.g., by acquiring completelyindividual characters, as occurs in progressive diversification ofembryonic cells and tissues. Cellular differentiation is a complexprocess typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell which itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typeseach can give rise to may vary considerably. Some differentiated cellsalso have the capacity to give rise to cells of greater developmentalpotential. Such capacity may be natural or may be induced artificiallyupon treatment with various factors. In many biological instances, stemcells are also “multipotent” because they can produce progeny of morethan one distinct cell type, but this is not required for “stem-ness.”Self-renewal is the other classical part of the stem cell definition,and it is essential as used in this document. In theory, self-renewalcan occur by either of two major mechanisms. Stem cells may divideasymmetrically, with one daughter retaining the stem state and the otherdaughter expressing some distinct other specific function and phenotype.Alternatively, some of the stem cells in a population can dividesymmetrically into two stems, thus maintaining some stem cells in thepopulation as a whole, while other cells in the population give rise todifferentiated progeny only. Formally, it is possible that cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen “reverse” and re-express the stem cell phenotype, a term oftenreferred to as “dedifferentiation” or “reprogramming” or“retro-differentiation” by persons of ordinary skill in the art. As usedherein, the term “pluripotent stem cell” includes embryonic stem cells,induced pluripotent stem cells, placental stem cells, etc.

The term “pluripotent” as used herein can refer to a cell with thecapacity, under different conditions, to differentiate to more than onedifferentiated cell type, and preferably to differentiate to cell typescharacteristic of all three germ cell layers. Pluripotent cells arecharacterized primarily by their ability to differentiate to more thanone cell type, preferably to all three germ layers, using, for example,a nude mouse teratoma formation assay. Pluripotency is also evidenced bythe expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. It should benoted that simply culturing such cells does not, on its own, render thempluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as thatterm is defined herein) also have the characteristic of the capacity ofextended passaging without loss of growth potential, relative to primarycell parents, which generally have capacity for only a limited number ofdivisions in culture.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and can refer to a pluripotent stem cellartificially derived (e.g., induced or by complete reversal) from anon-pluripotent cell, typically an adult somatic cell, for example, byinducing a forced expression of one or more genes.

The term “phenotype” can refer to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The terms “subject,” “patient,” or “individual” are used interchangeablyherein, and can refer to an animal, for example, a human from whom cellscan be obtained and/or to whom treatment, including prophylactictreatment, with the cells as described herein, is provided. Fortreatment of those infections, conditions or disease states which arespecific for a specific animal such as a human subject, the term subjectcan refer to that specific animal. The “non-human animals” and“non-human mammals” as used interchangeably herein, includes mammalssuch as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, andnon-human primates. The term “subject” also encompasses any vertebrateincluding but not limited to mammals, reptiles, amphibians and fish.However, advantageously, the subject is a mammal such as a human, orother mammals such as a domesticated mammal, e.g., dog, cat, horse, andthe like, or production mammal, e.g. cow, sheep, pig, and the like.“Patient in need thereof” or “subject in need thereof” is referred toherein as a patient diagnosed with or suspected of having a disease ordisorder, for instance, but not restricted to diabetes.

“Administering” used herein can refer to providing one or morecompositions described herein to a patient or a subject. By way ofexample and not limitation, composition administration, e.g., injection,can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.)injection, intradermal (i.d.) injection, intraperitoneal (i.p.)injection, or intramuscular (i.m.) injection. One or more such routescan be employed. Parenteral administration can be, for example, by bolusinjection or by gradual perfusion over time. Alternatively, orconcurrently, administration can be by the oral route. Additionally,administration can also be by surgical deposition of a bolus or pelletof cells, or positioning of a medical device. In an embodiment, acomposition of the present disclosure can comprise engineered cells orhost cells expressing nucleic acid sequences described herein, or avector comprising at least one nucleic acid sequence described herein,in an amount that is effective to treat or prevent proliferativedisorders. A pharmaceutical composition can comprise the cell populationas described herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions can comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

The terms “treat,” “treating,” “treatment,” and their grammaticalequivalents, as applied to an isolated cell, include subjecting the cellto any kind of process or condition or performing any kind ofmanipulation or procedure on the cell. As applied to a subject, theterms can refer to providing medical or surgical attention, care, ormanagement to an individual. The individual is usually ill or injured,or at increased risk of becoming ill relative to an average member ofthe population and in need of such attention, care, or management.

As used herein, the term “treating” and “treatment” can refer toadministering to a subject an effective amount of a composition so thatthe subject as a reduction in at least one symptom of the disease or animprovement in the disease, for example, beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptoms, diminishment of extent of disease, stabilized (e.g., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. As used herein, the term “treatment” includesprophylaxis. Alternatively, treatment is “effective” if the progressionof a disease is reduced or halted. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already diagnosed with acardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health.

The term “therapeutically effective amount,” therapeutic amount”, or itsgrammatical equivalents can refer to an amount effective, at dosages andfor periods of time necessary, to achieve a desired therapeutic result.The therapeutically effective amount can vary according to factors suchas the disease state, age, sex, and weight of the individual and theability of a composition described herein to elicit a desired responsein one or more subjects. The precise amount of the compositions of thepresent disclosure to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject).

Alternatively, the pharmacologic and/or physiologic effect ofadministration of one or more compositions described herein to a patientor a subject of can be “prophylactic,” e.g., the effect completely orpartially prevents a disease or symptom thereof. A “prophylacticallyeffective amount” can refer to an amount effective, at dosages and forperiods of time necessary, to achieve a desired prophylactic result(e.g., prevention of disease onset).

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

i) X is at least 100;

ii) X is at least 200;

iii) X is at least about 100; and

iv) X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

i) X being administered on between day 1 and day 2;

ii) X being administered on between day 2 and day 3;

iii) X being administered on between about day 1 and day 2;

iv) X being administered on between about day 2 and day 3;

v) X being administered on between day 1 and about day 2;

vi) X being administered on between day 2 and about day 3;

vii) X being administered on between about day 1 and about day 2; and

viii) X being administered on between about day 2 and about day 3.

I. Overview

Implantation of islet cells can replace dead or dysfunctional β cells indiabetes patients and potentially cure diabetes. Provided herein arecompositions and methods to produce stem-cell-derived β cells to be usedfor implantation.

Aspects of the present disclosure relates to methods of differentiatinga primitive gut tube cell into a Pdx1-positive pancreatic β cell. Insome cases, the method comprises contacting the primitive gut tube cellwith a composition comprising a bone morphogenetic protein (BMP)signaling pathway inhibitor and a growth factor from transformationgrowth factor β (TGF-β) superfamily. In some cases, the compositionfurther comprises one or more additional differentiation factors, whichinclude, but not limited to, a growth factor from fibroblast growthfactor (FGF) family, a Sonic Hedgehog (SHE) pathway inhibitor, aretinoic acid (RA) signaling pathway activator, a protein kinase C (PKC)activator, and a Rho-associated protein kinase (ROCK) inhibitor.

In some cases, a method provided herein comprises generating apopulation of cells or cell cluster that comprises a Pdx1-positivepancreatic progenitor cell by contacting a population of cellscomprising a primitive gut tube cell with a first composition comprisinga BMP signaling pathway inhibitor and a growth factor from TGF-βsuperfamily, wherein the primitive gut tube cell is differentiated inthe Pdx1-positive, NKX6.1-positive pancreatic progenitor cell. In somecases, the contacting takes place for about 1 day, 2 days, or 3 days. Insome cases, the contacting takes place about 1 day. In some cases, theprimitive gut tube cell is differentiated into a Pdx1-positive,NKX6.1-negative pancreatic progenitor cell by contacting with acomposition comprising BMP signaling pathway inhibitor and a growthfactor from TGF-β superfamily. In some cases, the generating stepfurther comprises differentiating the Pdx1-positive, NKX6.1-negativepancreatic progenitor cell into a Pdx1-positive, NKX6.1-positivepancreatic progenitor cell by contacting the Pdx1-positive,NKX6.1-negative pancreatic progenitor cell with a second compositioncomprising one or more differentiation factors, which include, but notlimited to, a growth factor from TGF-β superfamily, a growth factor fromFGF family, a SHH pathway inhibitor, a RA signaling pathway activator,and a ROCK inhibitor. In some cases, the second composition does notcomprise BMP signaling pathway inhibitor.

In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at most about 30%, at most about25%, at most about 20%, at most about 15%, at most about 10%, at mostabout 5%, at most about 4%, at most about 3%, at most about 2%, or atmost about 1% CHGA-positive cells by differentiating a population ofcells comprising primitive gut tube cells into a population of cells orcell cluster comprising Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells. In some cases, the method provided herein can obtain apopulation of cells or cell cluster that comprises at most about at mostabout 25%, at most about 20%, at most about 15%, or at most about 10%CDX2-positive cells as measured by flow cytometry by differentiating apopulation of cells comprising primitive gut tube cells into apopulation of cells or cell cluster comprising Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodprovided herein can obtain a population of cells or cell cluster thatcomprises at most about 30% CHGA-positive cells and at most about 30%CDX2-positive cells by differentiating a population of cells comprisingprimitive gut tube cells into a population of cells or cell clustercomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells.In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at most about 20% CHGA-positivecells and at most about 5% CDX2-positive cells by differentiating apopulation of cells comprising primitive gut tube cells into apopulation of cells or cell cluster comprising Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodprovided herein can obtain a population of cells or cell cluster thatcomprises at most about 15% CHGA-positive cells and at most about 3%CDX2-positive cells by differentiating a population of cells comprisingprimitive gut tube cells into a population of cells or cell clustercomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells.

In some cases, the BMP signaling pathway inhibitor provided hereincomprises DMH-1, derivative, analogue, or variant thereof. In someembodiments, the BMP signaling pathway provided herein comprises DMH-1.In some embodiments, the method comprises contacting primitive gut tubecell with about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM,about 0.1 μM to about 1 μM, or about 0.15 μM to about 0.5 μM DMH-1. Insome embodiments, the method comprises contacting primitive gut tubecell with about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21,0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33,0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.42, 0.45, 0.48, 0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.2, 1.4, 1.5, 1.6,1.7, 1.8, 2.0, 4.0, 6.0, 8.0, or 10 μM. In some embodiments, the methodcomprises contacting primitive gut tube cell with about 0.25 μM. In somecases, the BMP signaling pathway inhibitor as used in differentiatingthe primitive gut tube cell does not comprise LDN193189.

In some cases, the methods provided herein comprise generating apopulation of cells or cell cluster that comprise a Pdx1-positive,NKX6.1-positive pancreatic progenitor cell by contacting a population ofcells comprising a primitive gut tube cell with DMH-1, or derivative,analogue, or variant thereof.

Without wishing to be bound to a particular theory, in some embodimentsof the methods disclosed herein, during differentiation of a primitivegut tube cell to a Pdx1-positive pancreatic progenitor cell, inhibitionof BMP signaling pathway can contribute to reduction in generation ofoff-target cells, for instance, cells of intestine lineage or cellspositive for CDX2 gene expression. On the other hand, in some cases,during differentiation of a primitive gut tube cell to a Pdx1-positivepancreatic progenitor cell or Pdx1-positive, NKX6.1-positive pancreaticprogenitor cell, activation of Type II receptor-mediated TGF-β signalingpathway can contribute to reduction of early induction of neurogenin 3(Ngn3) or chromogranin A (CHGA), which can, in some cases, lead togeneration of polyhormonal cells rather than mature SC-β cells, which,in some cases, are monohormonal, e.g., secreting only insulin, but notother pancreatic hormones like somatostatin or glucagon. There can becross-talk between BMP signaling pathway and TGF-β signaling pathway. Insome cases, an inhibitor of BMP signaling pathway can have side effect,for instance, blockage of, among others, Type II receptor-mediated TGF-βsignaling pathway. The inhibition of Type II receptor-mediated TGF-βsignaling pathway, as illustrated in FIG. 15, for instance by arelatively less selective BMP signaling pathway inhibitor, LDN193189,can lead to early NGN3/CHGA induction, thereby generation ofpolyhormonal cells. Without wishing to be bound by a certain theory, insome cases, use of a highly selective BMP signaling pathway inhibitor,for instance, DMH-1 or its derivate, analogue, or variant, can have lessinhibitory effect on Type II receptor-mediated TGF-β signaling pathway.In some other cases, without wishing to be bound to a particular theory,co-incubation with a growth factor from TGF-β superfamily together witha BMP signaling pathway inhibitor can result in selective inhibition ofBMP signaling pathway, while maintaining relatively high activationlevel of Type II receptor-mediated TGF-β signaling pathway. In somecases, co-incubation with a growth factor from TGF-β superfamilytogether with a BMP signaling pathway inhibitor result in reducedgeneration of off-target cells, e.g., CDX2-positive cells, as well asreduced generation of polyhormonal cells, for instance, as a result ofearly induction of NGN3 or CHGA in the cells differentiated from theprimitive gut tube cells.

Aspects of the present disclosure relates to a method of generatingpancreatic β cells, e.g., SC-β cells, which comprises differentiatingprogenitor cells (e.g., stem cells like iPSC cells, definitive endodermcells, primitive gut tube cells, Pdx1-positive pancreatic progenitorcells, NKX6.1-positive pancreatic progenitor cells, or insulin-positiveendocrine cells) in a xeno-free culture medium. A xeno-free medium forculturing cells and/or cell clusters of originated from an animal canhave no product from other animals. In some cases, a xeno-free mediumfor culturing human cells and/or cell clusters can have no products fromany non-human animals. For example, a xeno-free medium for culturinghuman cells and/or cell clusters can comprise human serum albumin (HSA)or human platelet lysate (PLT) instead of fetal bovine serum (FBS) orbovine serum albumin (BSA).

In some embodiments, a method provided herein comprises generatingpancreatic β cells, e.g., SC-β cells, by differentiating progenitorcells (e.g., stem cells like iPSC cells, definitive endoderm cells,primitive gut tube cells, Pdx1-positive pancreatic progenitor cells,NKX6.1-positive pancreatic progenitor cells, or insulin-positiveendocrine cells) in a culture medium lacking serum albumin. In somecases, a population of cells or cell cluster comprising pancreatic βcells generated by a method provided herein that does not use serumalbumin or uses HSA in the culture medium can have significantimprovement as compared to a population of cells or cell clustercomprising pancreatic β cells generated by an otherwise identical methodbut using BSA instead. The improvement can include higher percentage ofpancreatic β cells in the final cell population obtained, higher GSISresponses (e.g., more insulin release in response to glucose challenge),higher GSIS stimulation index, more homogeneity of distribution ofpancreatic β cells in the cell cluster generated, or any combinationthereof.

In some embodiments, a method provided herein comprises differentiatinga population of cells comprising a stem cell, e.g., a hES cell or iPScell, in a culture medium comprising human serum albumin (HSA). In somecases, the stem cell is differentiated into a definitive endoderm cell.In some embodiments, a method provided herein comprises differentiatinga population of cells comprising a definitive endoderm cell in a culturemedium comprising human serum albumin (HSA). In some cases, thedefinitive endoderm cell is differentiated into a primitive gut tubecell. In some embodiments, a method provided herein comprisesdifferentiating a population of cells comprising a primitive gut tubecell in a culture medium comprising human serum albumin (HSA). In somecases, the primitive gut tube cell is differentiated into aPdx1-positive pancreatic progenitor cell (e.g., Pdx1-positive,NKX6.1-negative pancreatic progenitor cell or Pdx1-positive,NKX6.1-positive pancreatic progenitor cell). In some embodiments, amethod provided herein comprises differentiating a population of cellscomprising a Pdx1-positive, NKX6.1-negative pancreatic progenitor cellin a culture medium comprising human serum albumin (HSA). In some case,the Pdx1-positive, NKX6.1-negative pancreatic progenitor cell isdifferentiated into a Pdx1-positive, NKX6.1-positive pancreaticprogenitor cell. In some embodiments, a method provided herein comprisesdifferentiating a population of cells comprising a Pdx1-positive,NKX6.1-positive pancreatic progenitor cell in a culture mediumcomprising human serum albumin (HSA). In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cell is differentiated into aninsulin-positive endocrine cell. In some embodiments, a method providedherein comprises differentiating a population of cells comprising aninsulin-positive endocrine cell in a culture medium comprising humanserum albumin (HSA). In some cases, the insulin-positive endocrine cellis differentiated into a pancreatic β cell, e.g., SC-β cell.

In some embodiments, the methods provided herein comprise use of culturemedium comprising about 0.001% (w/v) to about 5% (w/v), about 0.005%(w/v) to about 4% (w/v), about 0.01% (w/v) to about 3% (w/v), about0.02% (w/v) to about 2.5% (w/v), about 0.03% (w/v) to about 2% (w/v),about 0.04% (w/v) to about 1% (w/v), about 0.045% (w/v) to about 0.5%(w/v), or about 0.05% (w/v) to about 0.1% (w/v) HSA. In someembodiments, the methods provided herein comprise use of culture mediumcomprising about 0.001%, 0.002%, 0.0025%, 0.005%, 0.0075%, 0.01%,0.0125%, 0.015%, 0.0175%, 0.02%, 0.0225%, 0.025%, 0.0275%, 0.03%,0.0325%, 0.035%, 0.0375%, 0.04%, 0.0425%, 0.045%, 0.0475%, 0.05%,0.0525%, 0.055%, 0.575%, 0.06%, 0.0625%, 0.065%, 0.0675%, 0.07%,0.0725%, 0.075%, 0.0775%, 0.08%, 0.085%, 0.09%, 0.1%, 0.12%, 0.15%,0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%,2.5%, 3%, or 4%, 5% (w/v) HSA. The term “w/v” is short for percentage ofweight/volume or weight per volume. For instance, 1 mg HSA in 100 mLculture medium has a concentration of 1% (w/v).

In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, or at leastabout 95% Pdx1-positive cells by differentiating a population of cellscomprising primitive gut tube cells into a population of cells or cellcluster comprising Pdx1-positive, NKX6.1-negative pancreatic progenitorcells. In some cases, the method provided herein can obtain a populationof cells or cell cluster that comprises at most about 60%, at most about50%, at most about 40%, at most about 35%, at most about 30%, at mostabout 25%, at most about 22%, at most about 20%, at most about 18%, atmost about 15%, at most about 14%, at most about 13%, at most about 11%,at most about 12%, at most about 10%, or at most about 5% CDX2-positivecells by differentiating a population of cells comprising primitive guttube cells into a population of cells or cell cluster comprisingPdx1-positive, NKX6.1-negative pancreatic progenitor cells.

Aspects of the disclosure relate to compositions and methods forgenerating stem cell-derived β (SC-β) cells (e.g., mature pancreatic βcells) from at least one insulin-positive endocrine cell or a precursorthereof (e.g., iPS cells, hESCs, definitive endoderm cells, primitivegut tube cells, PDX1-positive pancreatic progenitor cells,PDX1-positive, NKX6.1-positive pancreatic progenitor cells,Ngn3-positive endocrine progenitor cells, etc.), and SC-β cells producedby those compositions and methods for use in cell therapies, assays(e.g., drug screening), and various methods of treatment.

In some aspects, the disclosure provides methods and criteria to selectstem cells for producing SC-β cells. In some embodiments, the stem cellis embryonic stem cell (ESC). In some embodiments, the stem cell is acell expressing Oct4. In some embodiments, the starting stem cellculture comprises a percentage of Oct4 expressing cells of at least 80%,at least 85%, at least 90%, at least 95%, or 100%.

In some aspects, the disclosure relates to methods of identification ofthe SC-β cells that are detectable based on morphological criteria,without the need to employ a selectable marker, as well as functionalcharacteristics, such as ability to express insulin, secrete insulin inresponse to one or more glucose challenges, exhibit a mature GSISresponse, and organize in islets in pancreas in vivo, and typically havesmall spindle like cells of about 9-15 nm diameter.

In some aspects, the disclosure relates to methods of identifying basalmedium components for stem cell differentiation. In some embodiments,the basal medium components can increase differentiation efficiency. Insome embodiments, the basal medium components can increase thepercentage of on-target progenitor cell population (e.g., SC-β cellpopulation). In some embodiments, the percentage of on-target progenitorcell population produced by the disclosed methods is from 60% to 70%,from 70% to 80%, from 80% to 90%, or from to 100%. In some embodiments,the percentage of SC-β cell population produced by the disclosed methodsis from 60% to 70%, from 70% to 80%, from 80% to 90%, or from to 100%.In some embodiments, the percentage of SC-β cell population produced bythe disclosed methods is c

In some aspects, the disclosure relates to compositions and methods ofproducing SC-β cells, resulting in 1.2 to 3 fold increase of percentageof SC-β cells compared with standard methods known in the art. In someembodiments, the fold increase is from 1.2 to 1.5, from 1.5 to 2, orfrom 2 to 2.5. The produced SC-β cells can exhibit activity comparableto endogenous (e.g. natural) β cells and exhibit stability aftercryopreservation.

In some aspects, the disclosure relates to methods of identifyingsignaling factor that can improve SC-β cell activity (e.g. the abilityto sense glucose and secrete insulin). Screening of candidate smallmolecules can be performed to identify useful signaling factors. Oneexample assay can be used to test signaling factors is the GSIS assay.In some embodiments, the SC-β cell exhibits a stimulation index of atleast 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, or 5.0 or greater in the presence of a signaling factor.

II. SC-β Cells

The SC-β cells of the disclosure share many characteristic features of βcells which are important for normal β cell function. In someembodiments, the SC-β cell exhibits a glucose stimulated insulinsecretion (GSIS) response in vitro. In some embodiments, the SC-β cellexhibits a GSIS response in vivo. In some embodiments, the SC-β cellexhibits in vitro and in vivo GSIS responses. In some embodiments, theGSIS responses resemble the GSIS responses of an endogenous maturepancreatic β cell. In some embodiments, the SC-β cell exhibits a GSISresponse to at least one glucose challenge. In some embodiments, theSC-β cell exhibits a GSIS response to at least two sequential glucosechallenges. In some embodiments, the SC-β cell exhibits a GSIS responseto at least three sequential glucose challenges. In some embodiments,the GSIS responses resemble the GSIS response of endogenous human isletsto multiple glucose challenges. In some embodiments, the GSIS responseis observed immediately upon transplanting the cell into a human oranimal. In some embodiments, the GSIS response is observed withinapproximately 24 hours of transplanting the cell into a human or animal.In some embodiments, the GSIS response is observed within approximatelyone week of transplanting the cell into a human or animal. In someembodiments, the GSIS response is observed within approximately twoweeks of transplanting the cell into a human or animal. In someembodiments, the stimulation index of the cell as characterized by theratio of insulin secreted in response to high glucose concentrationscompared to low glucose concentrations is similar to the stimulationindex of an endogenous mature pancreatic β cell. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 1. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 1. In some embodiments, the SC-β cell exhibits a stimulationindex of greater than 1.1. In some embodiments, the SC-β cell exhibits astimulation index of greater than or equal to 1.1. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 2. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 1. In some embodiments, the SC-β cell exhibits a stimulationindex of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, or 5.0 or greater.

Some embodiments of the present disclosure relate to cell compositions,such as cell cultures or cell populations, comprising SC-β cells,wherein the SC-β cells have been derived from at least oneinsulin-positive endocrine cell or a precursor thereof. In someembodiments, the cell compositions comprise insulin-positive endocrinecells. In some embodiments, the cell compositions compriseNKX6.1-pancreatic progenitor cells. In some embodiments, the cellcompositions comprise PDX1-pancreatic progenitor cells. In someembodiments, the cell compositions comprise primitive gut tube cells. Insome embodiments, the cell compositions comprise definitive endodermcells.

In accordance with certain embodiments, the chemically induced SC-βcells are mammalian cells, and in a preferred embodiment, such SC-βcells are puma SC-β cells. In some embodiments, the insulin-positiveendocrine cells have been derived from definitive endoderm cells e.g.human definitive endoderm stem cells. In accordance with certainembodiments, the chemically induced PDX1-positive pancreatic progenitorsare mammalian cells, and in a preferred embodiment, such PDX1-positivepancreatic progenitors are human PDX1-positive pancreatic progenitors.

Other embodiments of the present disclosure relate to compositions, suchas an isolated cell population or cell culture, comprising SC-β cellsproduced by the methods as disclosed herein. In some embodiments of thepresent disclosure relate to compositions, such as isolated cellpopulations or cell cultures, comprising chemically-induced SC-β cellsproduced by the methods as disclosed herein. In such embodiments, theSC-β cells comprise less than about 90%, less than about 85%, less thanabout 80%, less than about 75%, less than about 70%, less than about65%, less than about 60%, less than about 55%, less than about 50%, lessthan about 45%, less than about 40%, less than about 35%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 12%, less than about 10%, less than about 8%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2% or less than about 1% of the total cells in theSC-β cells population. In some embodiments, the composition comprises apopulation of SC-β cells which make up more than about 90% of the totalcells in the cell population, for example about at least 95%, or atleast 96%, or at least 97%, or at least 98% or at least about 99%, orabout at least 100% of the total cells in the cell population are SC-βcells.

Certain other embodiments of the present disclosure relate tocompositions, such as an isolated cell population or cell cultures,comprise a combination of SC-β cells and insulin-positive endocrinecells or precursors thereof from which the SC-β cells were derived. Insome embodiments, the insulin-positive endocrine cells from which theSC-β cells are derived comprise less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2% or less than about1% of the total cells in the isolated cell population or culture.

Additional embodiments of the present disclosure relate to compositions,such as isolated cell populations or cell cultures, produced by theprocesses described herein and which comprise chemically induced SC-βcells as the majority cell type. In some embodiments, the methods andprocesses described herein produces an isolated cell culture and/or cellpopulations comprising at least about 99%, at least about 98%, at leastabout 97%, at least about 96%, at least about 95%, at least about 94%,at least about 93%, at least about 92%, at least about 91%, at leastabout 90%, at least about 89%, at least about 88%, at least about 87%,at least about 86%, at least about 85%, at least about 84%, at leastabout 83%, at least about 82%, at least about 81%, at least about 80%,at least about 79%, at least about 78%, at least about 77%, at leastabout 76%, at least about 75%, at least about 74%, at least about 73%,at least about 72%, at least about 71%, at least about 70%, at leastabout 69%, at least about 68%, at least about 67%, at least about 66%,at least about 65%, at least about 64%, at least about 63%, at leastabout 62%, at least about 61%, at least about 60%, at least about 59%,at least about 58%, at least about 57%, at least about 56%, at leastabout 55%, at least about 54%, at least about 53%, at least about 52%,at least about 51% or at least about 50% SC-β cells.

In another embodiment, isolated cell populations or compositions ofcells (or cell cultures) comprise human SC-β cells. In otherembodiments, the methods and processes as described herein can produceisolated cell populations comprising at least about 50%, at least about45%, at least about 40%, at least about 35%, at least about 30%, atleast about 25%, at least about 24%, at least about 23%, at least about22%, at least about 21%, at least about 20%, at least about 19%, atleast about 18%, at least about 17%, at least about 16%, at least about15%, at least about 14%, at least about 13%, at least about 12%, atleast about 11%, at least about 10%, at least about 9%, at least about8%, at least about 7%, at least about 6%, at least about 5%, at leastabout 4%, at least about 3%, at least about 2% or at least about 1% SC-βcells. In preferred embodiments, isolated cell populations can comprisehuman SC-β cells. In some embodiments, the percentage of SC-β cells inthe cell cultures or populations is calculated without regard to thefeeder cells remaining in the culture.

Yet another aspect of the present disclosure relates to cell populationsor compositions of cells (or cell cultures) that comprise at least about50%, at least about 45%, at least about 40%, at least about 35%, atleast about 30%, at least about 25%, at least about 24%, at least about23%, at least about 22%, at least about 21%, at least about 20%, atleast about 19%, at least about 18%, at least about 17%, at least about16%, at least about 15%, at least about 14%, at least about 13%, atleast about 12%, at least about 11%, at least about 10%, at least about9%, at least about 8%, at least about 7%, at least about 6%, at leastabout 5%, at least about 4%, at least about 3%, at least about 2% or atleast about 1% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 20% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 40% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 50% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises about17.9% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cell populationor composition of cells as provided herein comprises about 41.5%NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 50.6%NKX6.1⁺/C-peptide⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 90%, at least about 89%, atleast about 88%, at least about 85%, at least about 80%, at least about75%, at least about 70%, at least about 65%, at least about 60%, atleast about 55%, at least about 50%, at least about 45%, at least about40%, at least about 35%, at least about 30%, at least about 25%, atleast about 24%, at least about 23%, at least about 22%, at least about21%, at least about 20%, at least about 19%, at least about 18%, atleast about 17%, at least about 16%, at least about 15%, at least about14%, at least about 13%, at least about 12%, at least about 11%, atleast about 10%, at least about 9%, at least about 8%, at least about7%, at least about 6%, at least about 5%, at least about 4%, at leastabout 3%, at least about 2% or at least about 1% NKX6.1⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises at least about 40% NKX6.1⁺ cells. In some embodiments,the cell population or composition of cells as provided herein comprisesat least about 60% NKX6.1⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 85% NKX6.1⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 36.9% NKX6.1⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 63.4% NKX6.1⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 89.5% NKX6.1⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 55%, at least about 50%, atleast about 45%, at least about 40%, at least about 35%, at least about30%, at least about 25%, at least about 24%, at least about 23%, atleast about 22%, at least about 21%, at least about 20%, at least about19%, at least about 18%, at least about 17%, at least about 16%, atleast about 15%, at least about 14%, at least about 13%, at least about12%, at least about 11%, at least about 10%, at least about 9%, at leastabout 8%, at least about 7%, at least about 6%, at least about 5%, atleast about 4%, at least about 3%, at least about 2% or at least about1% C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises at least about 30%C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises at least about 55%C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 26.8% C-peptide⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 57.7% C-peptide⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 55.2% C-peptide⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 99%, at least about 98%, atleast about 95%, at least about 90%, at least about 89%, at least about88%, at least about 85%, at least about 80%, at least about 75%, atleast about 70%, at least about 65%, at least about 60%, at least about55%, at least about 50%, at least about 45%, at least about 40%, atleast about 35%, at least about 30%, at least about 25%, at least about24%, at least about 23%, at least about 22%, at least about 21%, atleast about 20%, at least about 19%, at least about 18%, at least about17%, at least about 16%, at least about 15%, at least about 14%, atleast about 13%, at least about 12%, at least about 11%, at least about10%, at least about 9%, at least about 8%, at least about 7%, at leastabout 6%, at least about 5%, at least about 4%, at least about 3%, atleast about 2% or at least about 1% Chromogranin A (CHGA)⁺ cells. Insome embodiments, the cell population or composition of cells asprovided herein comprises at least about 40% CHGA⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises at least about 85% CHGA⁺ cells. In some embodiments,the cell population or composition of cells as provided herein comprisesat least about 95% CHGA⁺ cells. In some embodiments, the cell populationor composition of cells as provided herein comprises about 37.7% CHGA⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 87.5% CHGA⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 96.4% CHGA⁺ cells.

Still other embodiments of the present disclosure relate tocompositions, such as isolated cell populations or cell cultures,comprising mixtures of SC-β cells and insulin-positive endocrine cellsor precursors thereof from which they were differentiated from. Forexample, cell cultures or cell populations comprising at least about 5SC-β cells for about every 95 insulin-positive endocrine cells orprecursors thereof can be produced. In other embodiments, cell culturesor cell populations comprising at least about 95 SC-β cells for aboutevery 5 insulin-positive endocrine cells or precursors thereof can beproduced. Additionally, cell cultures or cell populations comprisingother ratios of SC-β cells to insulin-positive endocrine cells orprecursors thereof are contemplated. For example, compositionscomprising at least about 1 SC-β cell for about every 1,000,000, or atleast 100,000 cells, or at least 10,000 cells, or at least 1000 cells or500, or at least 250 or at least 100 or at least 10 insulin-positiveendocrine cells or precursors thereof can be produced.

In some aspects, the present disclosure provides a cell clustercomprising at least about 50% Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells, at most about 30%, 28%, 26%, 25%, 24%, 22%, 20%, 18%,16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% chromogranin A(CHGA)-positive cells, and at most about 30%, 28%, 26%, 25%, 24%, 22%,20%, 18%, 16%, or 15% CDX2-positive cells. In some cases, the cellcluster comprises at most about 20% the CDX2-positive, NKX6.1-positivecells. In some cases, the cell cluster comprises at most about 5% theCHGA-positive cells. In some embodiments, the cell cluster comprises atmost about 20% the CDX2-positive, NKX6.1-positive cells and at mostabout 5% the CHGA-positive cells. In some embodiments, the cell clustercomprises at least about 60%, 62%, 64%, 65%, 68%, 70%, 72%, 74%, 76%,78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, or 95% the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some embodiments, thecell cluster comprises at least about 65% the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells.

In some embodiments, the cell cluster comprising at least about 50%Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, at mostabout 30% chromogranin A (CHGA)-positive cells, and at most about 30%CDX2-positive cells can have particular functional features as comparedto a comparable cell cluster having more than about 30% chromogranin A(CHGA)-positive cells or more than about 30% CDX2-positive cells. Forinstance, in some cases, further differentiation of the cell clustercomprising at least about 50% Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells, at most about 30% chromogranin A (CHGA)-positivecells, and at most about 30% CDX2-positive cells results in a first cellcluster comprising non-native pancreatic β cells that has a higherglucose-stimulated insulin secretion (GSIS) stimulation index than asecond cell cluster comprising the non-native pancreatic β cellsdifferentiated from a comparable cell cluster comprising at least about50% the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, andmore than 30% the chromogranin A (CHGA)-positive cells or more than 30%the CDX2-positive cells as measured by flow cytometry.

In some aspects, the present disclosure provides a cell clustercomprising at least about 60%, 62%, 64%, 65%, 68%, 70%, 72%, 74%, 76%,78%, 80%, 82%, 84%, 86%, 88%, or 90% Pdx1-positive, NKX6.1-negativepancreatic progenitor cells and at most about 40%, 38%, 36%, 34%, 32%,30%, 28%, 26%, 25%, 24%, 22%, 20%, 18%, 16%, 15%, 14%, 12%, or 10%CDX2-positive cells. In some embodiments, the cell cluster comprises atleast about 85% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells. In some embodiments, the cell cluster comprises at most about 15%the CDX2-positive cells. In some cases, the cell cluster comprises atleast about 85% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells and at most about 15% the CDX2-positive cells.

In some embodiments, the cell cluster comprising at least about 60%Pdx1-positive, NKX6.1-negative pancreatic progenitor cells and at mostabout 40% CDX2-positive cells can have particular functional features ascompared to a comparable cell cluster having more than about 40%CDX2-positive cells. For instance, in some cases, furtherdifferentiation of the cell cluster comprising at least about 60%Pdx1-positive, NKX6.1-negative pancreatic progenitor cells and at mostabout 40% CDX2-positive cells results in a first cell cluster comprisingnon-native pancreatic β cells that has a higher glucose-stimulatedinsulin secretion (GSIS) stimulation index than a second cell clustercomprising the non-native pancreatic β cells differentiated from acomparable cell cluster comprising at least about 60% Pdx1-positive,NKX6.1-negative pancreatic progenitor cells and more than 40% theCDX2-positive cells as measured by flow cytometry.

In some aspects, the present disclosure provides a cell clustercomprising non-native pancreatic β cells. In some embodiments, the cellcluster disclosed herein is obtained from differentiation of primitivegut tube cells by contacting the primitive gut tube cells with a bonemorphogenetic protein (BMP) signaling pathway inhibitor and a growthfactor from transformation growth factor β (TGF-β) superfamily. In someembodiments, the cell cluster has a higher number of the non-nativepancreatic β cells per cubic micrometer as compared to a comparablesecond cell cluster obtained from differentiation of primitive gut tubecells without the contacting. In some embodiments, cell cluster has anat least about 1.1, 1.2, 1.3, 1.4, 1.5, or 1.6 fold higher number of thenon-native pancreatic β cells per cubic micrometer as compared to thecomparable second cell cluster.

In some cases, the cell cluster comprising non-native pancreatic β cellsdisclosed herein exhibits higher insulin secretion in response toglucose challenge as compared to a comparable cell cluster obtained fromdifferentiation of primitive gut tube cells without contacting with BMPsignaling pathway inhibitor or growth factor from TGF-β family. In someembodiments, the cell cluster exhibits at least about 1.2, 1.5, 2, 2.5,3, 3.5, 4, 4.5, or 5 fold higher an insulin secretion as compared to thecomparable second cell cluster. In some embodiments, the cell clusterexhibits a higher GSIS stimulation index as compared to the comparablesecond cell cluster. In some embodiments, the GSIS stimulation index ofthe cell cluster is at least about 1.2 fold, at least about 1.5 fold, atleast about 1.8 fold, at least about 2 fold, at least about 2.2 fold, atleast about 2.4 fold, at least about 2.8 fold, or at least about 3 foldhigher than that of the second cell cluster. In some embodiments, GSISstimulation index of the cell cluster is at least about 3 fold higherthan that of the second population. In some embodiments, GSISstimulation index is calculated as a ratio of insulin secretion inresponse to 20 mM glucose challenge to insulin secretion in response to2 mM glucose challenge. In some embodiments, the non-native pancreatic βcells exhibit an in vitro glucose-stimulated insulin secretion responsewhen exposed to a glucose challenge. In some cases, non-nativepancreatic β cells exhibit an insulin secretion in response to a firstconcentration of K⁺. In some embodiments, the cell cluster exhibits ahigher insulin secretion as compared to the comparable second cellcluster in response to a first concentration of K⁺. In some embodiments,cell cluster exhibits at least about 1.2 fold, at least about 1.5 fold,at least about 1.8 fold, at least about 2 fold, at least about 2.2 fold,at least about 2.4 fold, at least about 2.8 fold, at least about 3 fold,at least about 3.2 fold, at least about 3.4 fold, at least about 3.6fold, at least about 3.8 fold, at least about 4 fold higher an insulinsecretion as compared to the comparable second cell cluster in responseto a first concentration of K⁺.

In some cases, cell populations or cell clusters disclosed herein areunsorted, e.g., isolated cell populations or cell clusters that have notbeen through cell sorting process. In some embodiments, the cell clusterdisclosed herein can refer to a cell cluster formed by self-aggregationof cells cultured in a given environment, for instance, in a 3Dsuspension culture. In some embodiments, cell clusters disclosed hereinare intermediate cell clusters formed during the differentiation processas described herein. In some cases, the intermediate cell clusters,e.g., cell clusters comprising Pdx1-positive, NKX6.1-negative pancreaticprogenitor cells (e.g., Stage 3 cell clusters) or cell clusterscomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells(e.g., Stage 4 cell clusters), are not subjected to cell sorting. Insome case, cell populations going through cell sorting may not be ableto form the intermediate cell clusters disclosed herein. For instance,Pdx1-positive pancreatic progenitor cells, after going through cellsorting, may not be able to form a cell cluster as disclosed herein.

Cell sorting as described herein can refer to a process of isolating agroup of cells from a plurality of cells by relying on differences incell size, shape (morphology), surface protein expression, endogenoussignal protein expression, or any combination thereof. In some cases,cell sorting comprises subjecting the cells to flow cytometry. Flowcytometry can be a laser- or impedance-based, biophysical technology.During flow cytometry, one can suspend cells in a stream of fluid andpass them through an electronic detection apparatus. In one type of flowcytometry, fluorescent-activated cell sorting (FACS), based on one ormore parameters of the cells' optical properties (e.g., emission wavelength upon laser excitation), one can physically separate and therebypurify cells of interest using flow cytometry. As described herein, anunsorted cell cluster can be cell cluster that formed by a plurality ofcells that have not been subject to an active cell sorting process,e.g., flow cytometry. In some cases, flow cytometry as discussed hereincan be based on one or more signal peptides expressed in the cells. Forexample, a cell cluster can comprise cells that express a signal peptide(e.g., a fluorescent protein, e.g., green fluorescent protein (GFP) ortdTomato). In some cases, the signal peptide is expressed as anindicator of insulin expression in the cells. For instance, a cellcluster can comprise cell harboring an exogenous nucleic acid sequencecoding for GFP under the control of an insulin promoter. The insulinpromoter can be an endogenous or exogenous promoter. In some cases, theexpression of GFP in these cells can be indicative of insulin expressionin the cells. The GFP signal can thus be a marker of a pancreatic βcell. In some cases, cell sorting as described herein can comprisemagnetic-activated flow cytometry, where magnetic antibody or otherligand is used to label cells of different types, and the differences inmagnetic properties can be used for cell sorting.

The percentage of cells expressing one or more particular markers, likePdx1, NKX6.1, insulin, NGN3, or CHGA, described herein can be thepercentage value detected using techniques like flow cytometry assay. Insome cases, during a flow cytometry assay, cell population or cellcluster discussed herein are dispersed into single-cell suspension byincubation in digesting enzyme like trypsin or TrypLE Express. Dispersedcell can be washed in suitable buffer like PBS, centrifuged and thenre-suspended in fixation buffer like 4% PFA. Incubation with primaryantibodies against the cell markers of interest can then be conducted,which can be followed by incubation with the secondary antibodies. Afterantibody incubation, the cells can be washed and the subject tosegregation by flow cytometry. Techniques other than flow cytometry canalso be used to characterize the cells described herein, e.g., determinethe cell percentages. Non-limiting examples of cell characterizationmethods include gene sequencing, microscopic techniques (fluorescencemicroscopy, atomic force microscopy), karyotyping, isoenzyme analysis,DNA properties, viral susceptibility.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl (e.g.,about 20 to about 50 mM, e.g., about 30 mM) as compared to the amount ofinsulin secreted upon induction with glucose. In some embodiments, thepopulation of glucose-responsive insulin secreting cells secrete atleast 1.5 times, 2 times, 2.5 times, 3 times higher amount of insulinupon induction with KCl as compared to the amount of insulin secretedupon induction with glucose.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl and/orglucose, in the presence of a signaling factor as compared to comparablecells in the absence of the signaling factor. In some embodiments, thecells secrete higher amount of insulin in the presence of high glucose,but not in the presence of low glucose. In some embodiments, the highglucose concentration is about 10-20 mM. In some embodiments, the lowglucose concentration is about 2-5 mM.

In some aspects, the disclosure relates to a composition comprising apopulation of differentiated pancreatic progenitor cells, wherein thepopulation comprises at least 60% pancreatic β cells as determined byflow cytometry. In some embodiments, the population comprises at least65%, 70%, 75%, 80%, 85%, or 90% pancreatic β cells. In some embodiments,the population comprises a higher percentage of pancreatic β cells uponbeing contacted with a predetermined basal medium component as comparedto a comparable population not contacted with the basal mediumcomponent.

The in vitro-matured, SC-β cell (e.g., pancreatic β cells) generatedaccording to the disclosed methods described herein demonstrate manyadvantages, for example, they perform glucose stimulated insulinsecretion in vitro, resemble human islet β cells by gene expression andultrastructure, secrete human insulin and ameliorate hyperglycemia whentransplanted into mice, provide a new platform for cell therapy (e.g.,transplantation into a subject in need of additional and/or functional βcells), drug screening (e.g., for insulin production/secretion,survival, dedifferentiation, etc.), research (e.g., determining thedifferences in function between normal and diabetic β cell), and tissueengineering (e.g., using the SC-β cells as the first cell type inreconstructing an islet).

III. Stem Cells and Reprogramming

Provided herein is use of stem cells for producing SC-β cells (e.g.,mature pancreatic β cells or (3-like cells) or precursors thereof. In anembodiment, germ cells may be used in place of, or with, the stem cellsto provide at least one SC-β cell, using similar protocols as describedin U.S. Patent Application Publication No. US20150240212 andUS20150218522, each of which is herein incorporated by reference in itsentirety. Suitable germ cells can be prepared, for example, fromprimordial germ cells present in human fetal material taken about 8-11weeks after the last menstrual period. Illustrative germ cellpreparation methods are described, for example, in Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.

Provided herein are compositions and methods of generating SC-β cells(e.g., pancreatic β cells). Generally, the at least one SC-β cell orprecursor thereof, e.g., pancreatic progenitors produced according tothe methods disclosed herein can comprise a mixture or combination ofdifferent cells, e.g., for example a mixture of cells such as primitivegut tube cells, Pdx1-positive pancreatic progenitors, Pdx1-positive,NKX6-1-positive pancreatic progenitors, Ngn3-positive endocrineprogenitor cells, insulin-positive endocrine cell (e.g., (3-like cells),and/or other pluripotent or stem cells.

The at least one SC-β cell or precursor thereof can be producedaccording to any suitable culturing protocol to differentiate a stemcell or pluripotent cell to a desired stage of differentiation. In someembodiments, the at least one SC-β cell or the precursor thereof areproduced by culturing at least one pluripotent cell for a period of timeand under conditions suitable for the at least one pluripotent cell todifferentiate into the at least one SC-β cell or the precursor thereof.

In some embodiments, the at least one SC-β cell or precursor thereof isa substantially pure population of SC-β cells or precursors thereof. Insome embodiments, a population of SC-β cells or precursors thereofcomprises a mixture of pluripotent cells or differentiated cells. Insome embodiments, a population SC-β cells or precursors thereof aresubstantially free or devoid of embryonic stem cells or pluripotentcells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one SC-β cell orprecursor thereof for use in the compositions and methods describedherein. In some embodiments, a somatic cell, e.g., fibroblast ismaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

In some embodiments, the at least one SC-β cell or precursor thereof aremaintained in culture by methods known by one of ordinary skills in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

Further, at least one SC-β cell or precursor thereof, e.g., pancreaticprogenitor can be from any mammalian species, with non-limiting examplesincluding a murine, bovine, simian, porcine, equine, ovine, or humancell. For clarity and simplicity, the description of the methods hereinrefers to a mammalian at least one SC-β cell or precursor thereof but itshould be understood that all of the methods described herein can bereadily applied to other cell types of at least one SC-β cell orprecursor thereof. In some embodiments, the at least one SC-β cell orprecursor thereof is derived from a human individual.

Stem Cells

Embodiments of the present disclosure can related to use of stem cellsfor generation of pancreatic β cells or precursors thereof. The term“stem cell” as used herein can refer to a cell (e.g., plant stem cell,vertebrate stem cell) that has the ability both to self-renew and togenerate a differentiated cell type (Morrison et al., (1997) Cell88:287-298). In the context of cell ontogeny, the adjective“differentiated”, or “differentiating” is a relative term. A“differentiated cell” can be a cell that has progressed further down thedevelopmental pathway than the cell it is being compared with. Thus,pluripotent stem cells can differentiate into lineage-restrictedprogenitor cells (e.g., mesodermal stem cells), which in turn candifferentiate into cells that are further restricted (e.g., neuronprogenitors), which can differentiate into end-stage cells (e.g.,terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.),which play a characteristic role in a certain tissue type, and can orcannot retain the capacity to proliferate further. Stem cells can becharacterized by both the presence of specific markers (e.g., proteins,RNAs, etc.) and the absence of specific markers. Stem cells can also beidentified by functional assays both in vitro and in vivo, particularlyassays relating to the ability of stem cells to give rise to multipledifferentiated progeny. In an embodiment, the host cell is an adult stemcell, a somatic stem cell, a non-embryonic stem cell, an embryonic stemcell, hematopoietic stem cell, an include pluripotent stem cells, and atrophoblast stem cell.

Stem cells that can be used in the method provided herein can includepluripotent stem cells (PSCs). The term “pluripotent stem cell” or “PSC”as used herein can refer to a stem cell capable of producing all celltypes of the organism. Therefore, a PSC can give rise to cells of allgerm layers of the organism (e.g., the endoderm, mesoderm, and ectodermof a vertebrate). Pluripotent cells can be capable of forming teratomasand of contributing to ectoderm, mesoderm, or endoderm tissues in aliving organism. Pluripotent stem cells of plants can be capable ofgiving rise to all cell types of the plant (e.g., cells of the root,stem, leaves, etc.).

Embodiments of the present disclosure can related to use of PSCs forgeneration of pancreatic β cells or precursors thereof. PSCs of animalscan be derived in a number of different ways. For example, embryonicstem cells (ESCs) can be derived from the inner cell mass of an embryo(Thomson et. al, Science. 1998 Nov. 6; 282(5391):1145-7) whereas inducedpluripotent stem cells (iPSCs) can be derived from somatic cells(Takahashi et. al, Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et. al,Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21;318(5858):1917-20. Epub 2007 Nov. 20). Because the term PSC can refer topluripotent stem cells regardless of their derivation, the term PSC canencompass the terms ESC and iPSC, as well as the term embryonic germstem cells (EGSC), which are another example of a PSC. PSCs can be inthe form of an established cell line, they can be obtained directly fromprimary embryonic tissue, or they can be derived from a somatic cell.

Embodiments of the present disclosure can related to use of ESCs forgeneration of pancreatic β cells or precursors thereof. By “embryonicstem cell” (ESC) can be meant a PSC that is isolated from an embryo,typically from the inner cell mass of the blastocyst. ESC lines arelisted in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01,hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3,HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMediHospital-Seoul National University); HSF-1, HSF-6 (University ofCalifornia at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin AlumniResearch Foundation (WiCell Research Institute)). Stem cells of interestalso include embryonic stem cells from other primates, such as Rhesusstem cells and marmoset stem cells. The stem cells can be obtained fromany mammalian species, e.g. human, equine, bovine, porcine, canine,feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al.(1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs can grow asflat colonies with large nucleo-cytoplasmic ratios, defined borders andprominent nucleoli. In addition, ESCs can express SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examplesof methods of generating and characterizing ESCs can be found in, forexample, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, each ofwhich is incorporated herein by its entirety. Methods for proliferatinghESCs in the undifferentiated form are described in WO 99/20741, WO01/51616, and WO 03/020920, each of which is incorporated herein by itsentirety.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EGcell”, it can be meant a PSC that is derived from germ cells and/or germcell progenitors, e.g. primordial germ cells, e.g. those that can becomesperm and eggs. Embryonic germ cells (EG cells) are thought to haveproperties similar to embryonic stem cells as described above. Examplesof methods of generating and characterizing EG cells may be found in,for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; andKoshimizu, U., et al. (1996) Development, 122:1235, each of which areincorporated herein by its entirety.

Embodiments of the present disclosure can related to use of iPSCs forgeneration of pancreatic β cells or precursors thereof. By “inducedpluripotent stem cell” or “iPSC”, it can be meant a PSC that is derivedfrom a cell that is not a PSC (e.g., from a cell this is differentiatedrelative to a PSC). iPSCs can be derived from multiple different celltypes, including terminally differentiated cells. iPSCs can have an EScell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nuclei. Inaddition, iPSCs can express one or more key pluripotency markers knownby one of ordinary skill in the art, including but not limited toAlkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181,TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples ofmethods of generating and characterizing iPSCs can be found in, forexample, U.S. Patent Publication Nos. US20090047263, US20090068742,US20090191159, US20090227032, US20090246875, and US20090304646, each ofwhich are incorporated herein by its entirety. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

Embodiments of the present disclosure can related to use of somaticcells for generation of pancreatic β cells or precursors thereof. By“somatic cell”, it can be meant any cell in an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells can becells that have differentiated sufficiently that they may not naturallygenerate cells of all three germ layers of the body, e.g. ectoderm,mesoderm and endoderm. For example, somatic cells can include bothneurons and neural progenitors, the latter of which is able to naturallygive rise to all or some cell types of the central nervous system butcannot give rise to cells of the mesoderm or endoderm lineages

In certain examples, the stem cells can be undifferentiated (e.g. a cellnot committed to a specific lineage) prior to exposure to at least onedifferentiation factor or composition according to the methods asdisclosed herein, whereas in other examples it can be desirable todifferentiate the stem cells to one or more intermediate cell typesprior to exposure of the at least one differentiation factor orcomposition described herein. In certain examples, the stem cells can becultured in the presence of) suitable nutrients and optionally othercells such that the stem cells can grow and optionally differentiate.For example, embryonic fibroblasts or fibroblast-like cells can bepresent in the culture to assist in the growth of the stem cells. Thefibroblast can be present during one stage of stem cell growth but notnecessarily at all stages. For example, the fibroblast can be added tostem cell cultures in a first culturing stage and not added to the stemcell cultures in one or more subsequent culturing stages.

Stem cells used in all aspects of the present invention can be any cellsderived from any kind of tissue (for example embryonic tissue such asfetal or pre-fetal tissue, or adult tissue), which stem cells can havethe characteristic of being capable under appropriate conditions ofproducing progeny of different cell types, e.g. derivatives of all of atleast one of the 3 germinal layers (endoderm, mesoderm, and ectoderm).These cell types can be provided in the form of an established cellline, or they can be obtained directly from primary embryonic tissue andused immediately for differentiation. Included are cells listed in theNIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02,hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-SeoulNational University); HSF-1, FISF-6 (University of California at SanFrancisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni ResearchFoundation (WiCell Research Institute)). In some embodiments, the sourceof human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdid not involve destroying a human embryo. In some embodiments, thesource of human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdo not involve destroying a human embryo.

In another example, the stem cells can be isolated from tissue includingsolid tissue. In some embodiments, the tissue is skin, fat tissue (e.g.adipose tissue), muscle tissue, heart or cardiac tissue. In otherembodiments, the tissue is for example but not limited to, umbilicalcord blood, placenta, bone marrow, or chondral.

Stem cells that can be used in the methods provided herein can alsoinclude embryonic cells of various types, exemplified by human embryonicstem (hES) cells, as described by Thomson et al, (1998) Science282:1145; embryonic stem cells from other primates, such as Rhesus stemcells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844);marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); andhuman embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998). Also applicable to the methods provided hereincan be lineage committed stem cells, such as mesodermal stem cells andother early cardiogenic cells (see Reyes et al, (2001) Blood98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16; etc.) Thestem cells can be obtained from any mammalian species, e.g. human,equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats,hamster, primate, etc. In some embodiments, a human embryo was notdestroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein. In some embodiments, a human embryo isnot destroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein.

A mixture of cells from a suitable source of endothelial, muscle, and/orneural stem cells can be harvested from a mammalian donor for thepurpose of the present disclosure. A suitable source is thehematopoietic microenvironment. For example, circulating peripheralblood, preferably mobilized (e.g., recruited), may be removed from asubject. In an embodiment, the stem cells can be reprogrammed stemcells, such as stem cells derived from somatic or differentiated cells.In such an embodiment, the de-differentiated stem cells can be forexample, but not limited to, neoplastic cells, tumor cells and cancercells or alternatively induced reprogrammed cells such as inducedpluripotent stem cells or iPS cells.

In some embodiments, the pancreatic β cell as described herein can bederived from one or more of trichocytes, keratinocytes, gonadotropes,corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells,parafollicular cells, glomus cells melanocytes, nevus cells, Merkelcells, odontoblasts, cementoblasts corneal keratocytes, retina Mullercells, retinal pigment epithelium cells, neurons, glias (e.g.,oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes(e.g., type I pneumocytes, and type II pneumocytes), clara cells, gobletcells, G cells, D cells, ECL cells, gastric chief cells, parietal cells,foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells,enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g.,Kupffer cells from mesoderm), cholecystocytes, centroacinar cells,pancreatic stellate cells, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic Ecells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroidchief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes,chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts,myocytes, myosatellite cells, tendon cells, cardiac muscle cells,lipoblasts, adipocytes, interstitial cells of cajal, angioblasts,endothelial cells, mesangial cells (e.g., intraglomerular mesangialcells and extraglomerular mesangial cells), juxtaglomerular cells,macula densa cells, stromal cells, interstitial cells, telocytes simpleepithelial cells, podocytes, kidney proximal tubule brush border cells,sertoli cells, leydig cells, granulosa cells, peg cells, germ cells,spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitorcells, endothelial stem cells, angioblasts, mesoangioblasts, pericytemural cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendriticcells, microphages, leukocytes), trophoblast stem cells, or anycombination thereof.

Reprogramming

The term “reprogramming” as used herein can refer to the process thatalters or reverses the differentiation state of a somatic cell. The cellcan either be partially or terminally differentiated prior to thereprogramming. Reprogramming can encompass complete reversion of thedifferentiation state of a somatic cell to a pluripotent cell. Suchcomplete reversal of differentiation can produce an induced pluripotent(iPS) cell. Reprogramming as used herein can also encompass partialreversion of a cells differentiation state, for example to a multipotentstate or to a somatic cell that is neither pluripotent or multipotent,but is a cell that has lost one or more specific characteristics of thedifferentiated cell from which it arises, e.g. direct reprogramming of adifferentiated cell to a different somatic cell type. Reprogramming caninvolve alteration, e.g., reversal, of at least some of the heritablepatterns of nucleic acid modification (e.g., methylation), chromatincondensation, epigenetic changes, genomic imprinting, etc., that occurduring cellular differentiation as a zygote develops into an adult.

As used herein, the term “reprogramming factor” can refer to a moleculethat is associated with cell “reprogramming,” that is, differentiation,and/or de-differentiation, and/or transdifferentiation, such that a cellconverts to a different cell type or phenotype. Reprogramming factorsgenerally affect expression of genes associated with celldifferentiation, de-differentiation and/or transdifferentiation.Transcription factors are examples of reprogramming factors.

The term “differentiation” and their grammatical equivalents as usedherein can refer to the process by which a less specialized cell (e.g.,a more naive cell with a higher cell potency) becomes a more specializedcell type (e.g., a less naive cell with a lower cell potency); and thatthe term “de-differentiation” can refer to the process by which a morespecialized cell becomes a less specialized cell type (e.g., a morenaive cell with a higher cell potency).

In some embodiments of the present disclosure, the method excludes theuse of reprogramming factor(s) that are not small molecules. However, itwill be appreciated that the method can utilize “routine” tissue culturecomponents such as culture media, serum, serum substitutes, supplements,antibiotics, etc, such as RPMI, Renal Epithelial Basal Medium (REBM),Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, CMRL 1066medium, F12, foetal calf serum (FCS), foetal bovine serum (FBS), bovineserum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (dipeptide,L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin,nicotinamide, insulin, transferrin, sodium selenite, selenium,ethanolamine, human epidermal growth factor (hEGF), basic fibroblastgrowth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin,streptomycin, gentamicin and amphotericin, etc. It is to be understoodthat these typical tissue culture components (and other similar tissueculture components that are routinely used in tissue culture) are notsmall molecule reprogramming molecules for the purposes of the presentdisclosure. These components are either not small molecules as definedherein and/or are not reprogramming factors as defined herein.

Accordingly, in an embodiment, the present disclosure does not involve aculturing step of the cell(s) with one or more exogenous polynucleotideor polypeptide reprogramming factor(s). Accordingly, in an embodiment,the method of the present disclosure does not involve the introductionof one or more exogenous polynucleotide or polypeptide reprogrammingfactor(s), e.g., by introducing transposons, viral transgenic vectors(such as retroviral vectors), plasmids, mRNA, miRNA, peptides, orfragments of any of these molecules, that are involved in producinginduced β cells or, otherwise, inducing cells of the present disclosureto differentiate, de-differentiation and/or transdifferentiate.

That is, in an embodiment, the method occurs in the absence of one ormore exogenous polynucleotide or polypeptide reprogramming factor(s).Accordingly, it is to be understood that in an embodiment, the method ofthe present disclosure utilizes small molecules (e.g., HDAC inhibitors)to reprogram cells, without the addition of polypeptide transcriptionfactors; other polypeptide factors specifically associated with inducingdifferentiation, de-differentiation, and/or transdifferentiation;polynucleotide sequences encoding polypeptide transcription factors,polynucleotide sequences encoding other polypeptide factors specificallyassociated with inducing differentiation, de-differentiation, and/ortransdifferentiation; mRNA; interference RNA; microRNA and fragmentsthereof.

IV Method of Generating Pancreatic β Cells

In some cases, the pancreatic β cells (e.g., non-native pancreatic βcells or SC-β cells) or the cell clusters comprising pancreatic β cellsas described herein are generated from any starting cell population invitro. For example, the starting cell can include, without limitation,insulin-positive endocrine cells (e.g., Ngn3-positive endocrine cells)or any precursor thereof, such as a Nkx6.1-positive pancreaticprogenitor cell, a Pdx1-positive pancreatic progenitor cell, a primitivegut tub cell, a definitive endoderm cell, a pluripotent stem cell, anembryonic stem cell, and an induced pluripotent stem cell. In somecases, the method includes differentiation of a reprogrammed cell, apartially reprogrammed cell (e.g., a somatic cell, e.g., a fibroblastwhich has been partially reprogrammed such that it exists in anintermediate state between an induced pluripotency cell and the somaticcell from which it has been derived), a transdifferentiated cell. Insome cases, the pancreatic β cell or the cell cluster comprising thepancreatic β cell disclosed herein can be differentiated in vitro froman insulin-positive endocrine cell or a precursor thereof. In somecases, the pancreatic β cell or the cell cluster comprising thepancreatic β cell is differentiated in vitro from a NKX6.1-positivepancreatic progenitor cell. In some cases, the pancreatic β cell or thecell cluster comprising the pancreatic β cell is differentiated in vitrofrom a Pdx1-positive pancreatic progenitor cell. In some cases, thepancreatic β cell or the cell cluster comprising the pancreatic β cellis differentiated in vitro from a primitive gut tube cell. In somecases, the pancreatic β cell or the cell cluster comprising thepancreatic β cell is differentiated in vitro from a definitive endodermcell. In some cases, the pancreatic β cell or the cell clustercomprising the pancreatic β cell is differentiated in vitro from apluripotent stem cell. In some cases, the pluripotent stem cell isselected from the group consisting of an embryonic stem cell and inducedpluripotent stem cell. As discussed above, the non-native pancreatic βcells can also be referred to as stem cell-derived β cells (SC-β cells)as they can be derived from stem cells in vitro. In some cases, the SC-βcell or the pluripotent stem cell from which the SC-β cell is derived ishuman. In some cases, the SC-β cell is human.

One aspect of the present disclosure relates to a method of propagatingstem cells, e.g., ES cells or pluripotent stem cells, e.g., iPS cells.In some cases, the stem cells can be cultured and propagated in asuitable culture medium, such as, RPMI, Renal Epithelial Basal Medium(REBM), Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, or CMRL1066 medium.

Some aspects of the present disclosure provide a method of generatingpancreatic β cells, e.g., non-native pancreatic β cells, or cell clustercomprising pancreatic β cells. In some cases, the method can be anycurrently available protocol, such as those described in U.S. patentapplication Ser. Nos. 14/684,129 and 14/684,101, each of which isincorporated herein by its entirety.

Aspects of the disclosure involve definitive endoderm cells. Definitiveendoderm cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, pluripotentstem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells.In some aspects, the endoderm cells (stage 1) are furtherdifferentiated, e.g., to primitive gut tube cells (stage 2),Pdx1-positive pancreatic progenitor cells (stage 3), NKX6.1-positivepancreatic progenitor cells (stage 4), or Ngn3-positive endocrineprogenitor cells or insulin-positive endocrine cells (stage 5), followedby induction or maturation to SC-β cells (stage 6).

In some cases, definitive endoderm cells can be obtained bydifferentiating at least some pluripotent cells in a population intodefinitive endoderm cells, e.g., by contacting a population ofpluripotent cells with i) at least one growth factor from the TGF-βsuperfamily, and ii) a WNT signaling pathway activator, to induce thedifferentiation of at least some of the pluripotent cells intodefinitive endoderm cells, wherein the definitive endoderm cells expressat least one marker characteristic of definitive endoderm.

Any growth factor from the TGF-β superfamily capable of inducing thepluripotent stem cells to differentiate into definitive endoderm cells(e.g., alone, or in combination with a WNT signaling pathway activator)can be used in the method provided herein. In some cases, the growthfactor from the TGF-β superfamily comprises Activin A. In some cases,the growth factor from the TGF-β superfamily comprises growthdifferentiating factor 8 (GDF8). Any WNT signaling pathway activatorcapable of inducing the pluripotent stem cells to differentiate intodefinitive endoderm cells (e.g., alone, or in combination with a growthfactor from the TGF-β superfamily) can be used in the method providedherein. In some cases, the WNT signaling pathway activator comprisesCHIR99Q21. In some cases, the WNT signaling pathway activator comprisesWnt3a recombinant protein.

In some cases, differentiating at least some pluripotent cells in apopulation into definitive endoderm cells is achieved by a process ofcontacting a population of pluripotent cells with i) Activin A, and ii)CHIR99021 for a suitable period of time, e.g., about 2 days, about 3days, about 4 days, or about 5 days to induce the differentiation of atleast some of the pluripotent cells in the population into definitiveendoderm cells, wherein the definitive endoderm cells express at leastone marker characteristic of definitive endoderm.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the growth factor from the TGF-βsuperfamily (e.g., Activin A), such as, about 10 ng/mL, about 20 ng/mL,about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In somecases, the method comprises use of about 100 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 200 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the WNT signaling pathwayactivator (e.g., CHIR99021), such as, about 0.01 μM, about 0.05 μM,about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM,about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM,about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. Insome cases, the method comprises use of about 2 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 5 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells.

In some cases, a definitive endoderm cell produced by the methods asdisclosed herein expresses at least one marker selected from the groupconsisting of: Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3g12, Ripk4,Rab1S, Npnt, Clic6, CldnS, Cacnalb, Bnipl, Anxa4, Emb, FoxA1, Sox17, andRbm35a, wherein the expression of at least one marker is upregulated toby a statistically significant amount in the definitive endoderm cellrelative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Gata4, SPARC, AFP and Dab2relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Zic1, Pax6, Flk1 and CD31relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein has a higher level of phosphorylation of Smad2 by a statisticallysignificant amount relative to the pluripotent stem cell from which itwas derived. In some cases, a definitive endoderm cell produced by themethods as disclosed herein has the capacity to form gut tube in vivo.In some cases, a definitive endoderm cell produced by the methods asdisclosed herein can differentiate into a cell with morphologycharacteristic of a gut cell, and wherein a cell with morphologycharacteristic of a gut cell expresses FoxA2 and/or Claudin6, In somecases, a definitive endoderm cell produced by the methods as disclosedherein can be further differentiated into a cell of endoderm origin.

In some cases, a population of pluripotent stem cells are cultured inthe presence of at least one β cell differentiation factor prior to anydifferentiation or during the first stage of differentiation. One canuse any pluripotent stem cell, such as a human pluripotent stem cell, ora human iPS cell or any of pluripotent stem cell as discussed herein orother suitable pluripotent stem cells. In some cases, a β celldifferentiation factor as described herein can be present in the culturemedium of a population of pluripotent stem cells or may be added inbolus or periodically during growth (e.g. replication or propagation) ofthe population of pluripotent stem cells. In certain examples, apopulation of pluripotent stem cells can be exposed to at least one βcell differentiation factor prior to any differentiation. In otherexamples, a population of pluripotent stem cells may be exposed to atleast one β cell differentiation factor during the first stage ofdifferentiation.

Aspects of the disclosure involve primitive gut tube cells. Primitivegut tube cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, definitiveendoderm cells are differentiated to primitive gut tube cells. In someaspects, the primitive gut tube cells are further differentiated, e.g.,to Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreaticprogenitor cells, Ngn3-positive endocrine progenitor cells,insulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with at least one growth factor from the fibroblast growth factor(FGF) family, to induce the differentiation of at least some of thedefinitive endoderm cells into primitive gut tube cells, wherein theprimitive gut tube cells express at least one marker characteristic ofprimitive gut tube cells.

Any growth factor from the FGF family capable of inducing definitiveendoderm cells to differentiate into primitive gut tube cells (e.g.,alone, or in combination with other factors) can be used in the methodprovided herein. In some cases, the at least one growth factor from theFGF family comprises keratinocyte growth factor (KGF). In some cases,the at least one growth factor from the FGF family comprises FGF2. Insome cases, the at least one growth factor from the FGF family comprisesFGF8B. In some cases, the at least one growth factor from the FGF familycomprises FGF 10. In some cases, the at least one growth factor from theFGF family comprises FGF21.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with KGF for a certain period of time, e.g., about 1 day, about 2days, about 3 days, or about 4 days, to induce the differentiation of atleast some of the definitive endoderm cells into primitive gut tubecells.

In some cases, the method comprises differentiating definitive endodermcells into primitive gut tube cells by contacting definitive endodermcells with a suitable concentration of the growth factor from the FGFfamily (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL,about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL,about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL,about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some cases, themethod comprises use of about 50 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells. In some cases,the method comprises use of about 100 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells.

Aspects of the disclosure involve Pdx1-positive pancreatic progenitorcells. Pdx1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, primitive gut tube cells are differentiatedto Pdx1-positive pancreatic progenitor cells. In some aspects, thePdx1-positive pancreatic progenitor cells are further differentiated,e.g., NKX6.1-positive pancreatic progenitor cells, Ngn3-positiveendocrine progenitor cells, insulin-positive endocrine cells, followedby induction or maturation to SC-β cells,

In some aspects, Pdx1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; vi) at least one protein kinase C activator, and vii) ROCKinhibitor to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

In some aspects, Pdx1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; and vi) at least one protein kinase C activator, to inducethe differentiation of at least some of the primitive gut tube cellsinto Pdx1-positive pancreatic progenitor cells, wherein thePdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) at least one growth factor from the FGF family,iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid(RA) signaling pathway activator; and v) at least one protein kinase Cactivator, to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one SHH pathwayinhibitor, ii) at least one retinoic acid (RA) signaling pathwayactivator; and iii) at least one protein kinase C activator, wherein thePdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one growth factorfrom the FGF family, and ii) at least one retinoic acid (RA) signalingpathway activator, to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

Any BMP signaling pathway inhibitor capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of a growth factor fromTGF-β superfamily, at least one growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used in the method provided herein. In some cases, theBMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of BMP signaling pathway inhibitor (e.g., LDN1931189),such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM,about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM,about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM,about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM,about 400 nM, about 500 nM, or about 1 μM. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration ofBMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 μM,about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM,about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM,about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM,about 40 μM, about 50 μM, or about 100 μM.

Any growth factor from the TGF-β superfamily capable of inducingprimitive gut tube cells to differentiate into Pdx1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least oneBMP signaling pathway inhibitor, a growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the growth factor from TGF-βfamily comprises Activin A. In some cases, the growth factor from TGF-βfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration of agrowth factor from TGF-13 superfamily (e.g., Activin A), such as, about5 ng/mL, about 7.5 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL,about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL,about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, or about 100 ng/mL.

Any growth factor from the FGF family capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, a growth factor from TGF-β superfamily, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the at least one growth factorfrom the FGF family comprises keratinocyte growth factor (KGF). In somecases, the at least one growth factor from the FGF family is selectedfrom the group consisting of FGF2, FGF8B, FGF 10, and FGF21. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of a growth factor from FGF family (e.g., KGF), such as,about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL,about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL,about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, orabout 300 ng/mL.

Any SHH pathway inhibitor capable of inducing primitive gut tube cellsto differentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, a growthfactor from TGF-β superfamily, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the SHH pathway inhibitorcomprises Sant1. In some examples, the method comprises contactingprimitive gut tube cells with a concentration of a SHH pathway inhibitor(e.g., Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM,about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM,about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM,about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, at least one growth factor from the FGFfamily, at least one SHH pathway inhibitor, at least one protein kinaseC activator, and ROCK inhibitor) can be used. In some cases, the RAsignaling pathway activator comprises retinoic acid. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any PKC activator capable of inducing primitive gut tube cells todifferentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, at least one RA signaling pathway activator, andROCK inhibitor) can be used. In some cases, the PKC activator comprisesPdBU. In some cases, the PKC activator comprises TPB. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of a PKC activator (e.g., PdBU), such as, about 10 μM,about 20 μM, about 50 μM, about 75 μM, about 80 μM, about 100 μM, about120 μM, about 140 μM, about 150 μM, about 175 μM, about 180 μM, about200 μM, about 210 μM, about 220 μM, about 240 μM, about 250 μM, about260 μM, about 280 μM, about 300 μM, about 320 μM, about 340 μM, about360 μM, about 380 μM, about 400 μM, about 420 μM, about 440 μM, about460 μM, about 480 μM, about 500 μM, about 520 μM, about 540 μM, about560 μM, about 580 μM, about 600 μM, about 620 μM, about 640 μM, about660 μM, about 680 μM, about 700 μM, about 750 μM, about 800 μM, about850 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, orabout 5 mM.

Any ROCK inhibitor capable of inducing primitive gut tube cells todifferentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, PKC activator, and at least one RA signalingpathway activator) can be used. In some cases, the ROCK inhibitorcomprises Y-27632, Fasudil/HA1077, or H-1152. In some cases, the ROCKinhibitor comprises Y-27632. In some examples, the method comprisescontacting primitive gut tube cells with a concentration of a ROCKinhibitor (e.g., Y-27632), such as, about 0.2 μM, about 0.5 μM, about0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM,about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM,about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM,about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about40 μM, about 50 μM, or about 100 μM.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with retinoic acid, KGF, Sant1,LDN193189, PdBU, Y-27632, and Activin A, for a suitable period of time,e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In somecases, Pdx1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in a populationinto Pdx1-positive pancreatic progenitor cells, e.g., by contactingprimitive gut tube cells with retinoic acid, KGF, Sant1, LDN193189,PdBU, Y-27632, and Activin A, for about 2 days. In some cases,Pdx1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in S3 medium.

Aspects of the disclosure involve NKX6.1-positive pancreatic progenitorcells. NKX6.1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, Pdx1-positive pancreatic progenitor cells aredifferentiated to NKX6.1-positive pancreatic progenitor cells. In someaspects, the NKX6.1-positive pancreatic progenitor cells are furtherdifferentiated, e.g., to Ngn3-positive endocrine progenitor cells, orinsulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some aspects, a method of producing a NKX6.1-positive pancreaticprogenitor cell from a Pdx1-positive pancreatic progenitor cellcomprises contacting a population of cells (e.g., under conditions thatpromote cell clustering and/or promoting cell survival) comprisingPdx1-positive pancreatic progenitor cells with at least two βcell-differentiation factors comprising a) at least one growth factorfrom the fibroblast growth factor (FGF) family, b) a sonic hedgehogpathway inhibitor, and optionally c) a low concentration of a retinoicacid (RA) signaling pathway activator, to induce the differentiation ofat least one Pdx1-positive pancreatic progenitor cell in the populationinto NKX6.1-positive pancreatic progenitor cells, wherein theNKX6.1-positive pancreatic progenitor cells expresses NKX6.1.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, to induce the differentiation of atleast some of the Pdx1-positive pancreatic progenitor cells intoPdx1-positive, NKX6.1-positive pancreatic progenitor cells, wherein thePdx1-positive, NKX6.1-positive pancreatic progenitor cells expressesPdx1 and NKX6.1.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, iv) ROCK inhibitor, and v) at leastone growth factor from the TGF-β superfamily, to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells. In some cases, the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells are obtained by contacting Pdx1-positivepancreatic progenitor cells under conditions that promote cellclustering with at least one growth factor from the FGF family.

In some cases, the Pdx1-positive pancreatic progenitor cells areproduced from a population of pluripotent cells. In some cases, thePdx1-positive pancreatic progenitor cells are produced from a populationof iPS cells. In some cases, the Pdx1-positive pancreatic progenitorcells are produced from a population of ESC cells. In some cases, thePdx1-positive pancreatic progenitor cells are produced from a populationof definitive endoderm cells. In some cases, the Pdx1-positivepancreatic progenitor cells are produced from a population of primitivegut tube cells.

Any growth factor from the FGF family capable of inducing Pdx1-positivepancreatic-progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one SHH pathway inhibitor, a ROCK inhibitor, a growth factor fromthe TGF-β superfamily, and at least one retinoic acid signaling pathwayactivator) can be used in the method provided herein. In some cases, theat least one growth factor from the FGF family comprises keratinocytegrowth factor (KGF). In some cases, the at least one growth factor fromthe FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method comprises contactingPdx1-positive pancreatic progenitor cells with a concentration of agrowth factor from FGF family (e.g., KGF), such as, about 10 ng/mL,about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300ng/mL.

Any SHH pathway inhibitor capable of inducing Pdx1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one retinoic acid signalingpathway activator, ROCK inhibitor, and at least one growth factor fromthe TGF-β superfamily) can be used in the method provided herein. Insome cases, the SHH pathway inhibitor comprises Sant1. In some examples,the method comprises contacting Pdx1-positive pancreatic progenitorcells with a concentration of a SHH pathway inhibitor (e.g., Sant1),such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM,about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.104,about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about0.2104, about 0.2204, about 0.23 μM, about 0.24 μM, about 0.25 μM, about0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM,about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing Pdx1-positivepancreatic progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one growth factor from the FGF family, at least one SHH pathwayinhibitor, ROCK inhibitor, and at least one growth factor from the TGF-βsuperfamily) can be used. In some cases, the RA signaling pathwayactivator comprises retinoic acid. In some examples, the methodcomprises contacting Pdx1-positive pancreatic progenitor cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.104, about 0.2 μM, about 0.25 μM,about 0.3 μM, about 0.4 μM, about 0.45 nM, about 0.5 nM, about 0.55 nM,about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 nM, about 0.8 nM,about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 nM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any ROCK inhibitor capable of inducing Pdx1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one SHH pathway inhibitor, aRA signaling pathway activator, and at least one growth factor from theTGF-β superfamily) can be used. In some cases, the ROCK inhibitorcomprises Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152. In someexamples, the method comprises contacting Pdx1-positive pancreaticprogenitor cells with a concentration of a ROCK inhibitor (e.g.,Y-27632), such as, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM,about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM,about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM,or about 100 μM.

Any activator from the TGF-β superfamily capable of inducingPdx1-positive pancreatic progenitor cells to differentiate intoNKX6.1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one growth factor from the FGF family, at leastone SHH pathway inhibitor, a RA signaling pathway activator, and ROCKinhibitor) can be used. In some cases, the activator from the TGF-βsuperfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting Pdx1-positive pancreatic progenitor cells with aconcentration of a growth factor from TGF-β superfamily (e.g., ActivinA), such as, about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8ng/mL, about 1 ng/mL, about 1.2 ng/mL, about 1.4 ng/mL, about 1.6 ng/mL,about 1.8 ng/mL, about 2 ng/mL, about 2.2 ng/mL, about 2.4 ng/mL, about2.6 ng/mL, about 2.8 ng/mL, about 3 ng/mL, about 3.2 ng/mL, about 3.4ng/mL, about 3.6 ng/mL, about 3.8 ng/mL, about 4 ng/mL, about 4.2 ng/mL,about 4.4 ng/mL, about 4.6 ng/mL, about 4.8 ng/mL, about 5 ng/mL, about5.2 ng/mL, about 5.4 ng/mL, about 5.6 ng/mL, about 5.8 ng/mL, about 6ng/mL, about 6.2 ng/mL, about 6.4 ng/mL, about 6.6 ng/mL, about 6.8ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 20 ng/mL, about 30 ng/mL, or about 50 ng/mL. In some examples, themethod comprises contacting Pdx1-positive pancreatic progenitor cellswith a concentration of a growth factor from TGF-β superfamily (e.g.,Activin A), such as, about 5 ng/mL.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells under conditions that promote cell clustering with KGF, Sant1, andRA, for a period of 5 days. In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells are obtained by contactingPdx1-positive pancreatic progenitor cells under conditions that promotecell clustering with KGF, Sant1, RA, Y27632, and Activin A, for a periodof 5 days. In some cases, the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells under conditions that promote cell clustering with KGFfor a period of 5 days. In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells are obtained by contactingPdx1-positive pancreatic progenitor cells in a S3 medium.

Aspects of the disclosure involve insulin-positive endocrine cells.Insulin-positive endocrine cells of use herein can be derived from anysource or generated in accordance with any suitable protocol, In someaspects, NKX6.1-positive pancreatic progenitor cells are differentiatedto insulin-positive endocrine cells, In some aspects, theinsulin-positive endocrine cells are further differentiated, e.g., byinduction or maturation to SC-β cells.

In some aspects, a method of producing an insulin-positive endocrinecell from an NKX6.1-positive pancreatic progenitor cell comprisescontacting a population of cells (e.g., under conditions that promotecell clustering) comprising NKX6-l-positive pancreatic progenitor cellswith a) a TGF-β signaling pathway inhibitor, and b) a thyroid hormonesignaling pathway activator, to induce the differentiation of at leastone NKX6.1-positive pancreatic progenitor cell in the population into aninsulin-positive endocrine cell, wherein the insulin-positive endocrineceil expresses insulin. In some cases, insulin-positive endocrine cellsexpress Pdx1, NKX6.1, NKX2.2, Math, glis3, Sur1, Kir6.2, Znt8, SLC2A1,SLC2A3 and/or insulin.

Any TGF-β signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other β cell-differentiation factors, e.g., a thyroidhormone signaling pathway activator) can be used. In some cases, theTGF-β signaling pathway comprises TGF-β receptor type I kinasesignaling. In some cases, the TGF-β signaling pathway inhibitorcomprises Alk5 inhibitor II.

Any thyroid hormone signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other β cell-differentiation factors, e.g., a TGF-βsignaling pathway inhibitor) can be used. In some cases, the thyroidhormone signaling pathway activator comprises triiodothyronine (T3).

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with at least oneadditional factor. In some cases, the method comprises contacting thePdx1-positive NKX6.1-positive pancreatic progenitor cells with at leastone of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator,iii) a γ-secretase inhibitor, iv) at least one growth factor from theepidermal growth factor (EGF) family, and optionally v) a protein kinaseinhibitor.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with at least oneadditional factor. In some cases, the method comprises contacting thePdx1-positive NKX6.1-positive pancreatic progenitor cells with at leastone of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator,iii) a γ-secretase inhibitor, iv) at least one growth factor from theepidermal growth factor (EGF) family, and v) at least one bonemorphogenetic protein (BMP) signaling pathway inhibitor.

Any γ-secretase inhibitor that is capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator). In some cases, theγ-secretase inhibitor comprises XXI. In some cases, the γ-secretaseinhibitor comprises DAPT. In some examples, the method comprisescontacting NKX6.1-positive pancreatic progenitor cells with aconcentration of a γ-secretase inhibitor (e.g., XXI), such as, about0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM,about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM,about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM,about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM,about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM,about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM,about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM.

Any growth factor from the EGF family capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the at least one growth factor from the EG F family comprisesbetacellulin. In some cases, at least one growth factor from the EGFfamily comprises EGF. In some examples, the method comprises contactingNKX6.1-positive pancreatic progenitor cells with a concentration of agrowth factor from EGF family (e.g., betacellulin), such as, about 1ng/mL, about 2 ng/mL, about 4 ng/mL, about 6 ng/mL, about 8 ng/mL, about10 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18ng/mL, about 20 ng/mL, about 22 ng/mL, about 24 ng/mL, about 26 ng/mL,about 28 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 75ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL,about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL.

Any RA signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the RA signaling pathway activator comprises RA. In some examples, themethod comprises contacting NKX6.1-positive pancreatic progenitor cellswith a concentration of an RA signaling pathway activator (e.g.,retinoic acid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM,about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM,about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM,about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM,about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM,about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any SHH pathway inhibitor capable of inducing the differentiation ofNKX6.1-positive pancreatic progenitor cells to differentiate intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator) can be used in the method provided herein.In some cases, the SHH pathway inhibitor comprises Sant1. In someexamples, the method comprises contacting NKX6.1-positive pancreaticprogenitor cells with a concentration of a SHH pathway inhibitor (e.g.,Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about0.01 μM, about 0.02 μM, about 0.03 μM, about 0.0504, about 0.08 μM,about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM,about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any BMP signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. Insome examples, the method comprises contacting NKX6.1-positivepancreatic progenitor cells with a concentration of BMP signalingpathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM,about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about1 μM.

In some cases, the population of cells is optionally contacted with aprotein kinase inhibitor. In some cases, the population of cells is notcontacted with the protein kinase inhibitor. In some cases, thepopulation of cells is contacted with the protein kinase inhibitor. Anyprotein kinase inhibitor that is capable of inducing the differentiationof NKX6.1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator). In some cases, the protein kinaseinhibitor comprises staurosporine.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3,RA, Sant1, and betacellulin for a period of 7 days, to induce thedifferentiation of at least one NKX6.1-positive pancreatic progenitorcell in the population into an insulin-positive endocrine cell, whereinthe insulin-positive endocrine cell expresses insulin. In some cases,the method comprises contacting the population of cells (e.g.,NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3, RA,Sant1, betacellulin, and LDN193189 for a period of 7 days, to induce thedifferentiation of at least one NKX6.1-positive pancreatic progenitorcell in the population into an insulin-positive endocrine cell, whereinthe insulin-positive endocrine ceil expresses insulin. In someembodiments, one or more differentiation factors are added in a portionof the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6 days ofthe period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6 days ofthe period of time for Stage 5. In one example, the cells are contactedwith SHH signaling pathway inhibitor for only the first 2, 3, 4, or 5days during Stage 5, after which the SHH signaling pathway inhibitor isremoved from the culture medium. In another example, the cells arecontacted with BMP signaling pathway inhibitor for only the first 1, 2,or 3 days during Stage 5, after which the BMP signaling pathwayinhibitor is removed from the culture medium.

In some cases, the method comprises culturing the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) in a BE5 medium, toinduce the differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin.

Aspects of the disclosure involve generating pancreatic β cells (e.g.,non-native pancreatic β cells). Non-native pancreatic β cells, in somecases, resemble endogenous mature β cells in form and function, butnevertheless are distinct from native β cells.

In some cases, the insulin-positive pancreatic endocrine cells generatedusing the method provided herein can form a cell cluster, alone ortogether with other types of cells, e.g., precursors thereof, e.g., stemcell, definitive endoderm cells, primitive gut tube cell, Pdx1-positivepancreatic progenitor cells, or NKX6.1-positive pancreatic progenitorcells.

In some cases, the cell population comprising the insulin-positiveendocrine cells can be directly induced to mature into SC-β cellswithout addition of any exogenous differentiation factors (such asinhibitor of TGF-β signaling pathway, thyroid hormone signaling pathwayactivator, PKC activator, growth factors from TGF-β superfamily, FGFfamily, or EGF family, SHH signaling pathway inhibitor, γ-secretaseinhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor).

In some cases, the cell population comprising the insulin-positiveendocrine cells can be directly induced to mature into SC-β cells bycontacting the insulin-positive endocrine cells with differentiationfactors. The differentiation factors can comprise at least one inhibitorof TGF-β signaling pathway and thyroid hormone signaling pathwayactivator as described herein. In some cases, SC-β cells can be obtainedby contacting a population of cells comprising insulin-positiveendocrine cells with Alk5i and T3.

In some examples, insulin-positive endocrine cells can be matured in aNS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In somecases, the insulin-positive endocrine cells can be matured in a CMRLsmedium supplemented with 10% FBS. In some cases, the insulin-positiveendocrine cells can be matured in a DMEM medium supplemented with 1%HSA. In other cases, SC-β cells can be obtained by culturing thepopulation of cells containing the insulin-positive endocrine cells in aMCDB131 medium that can be supplemented by 2% BSA. In some cases, theMCDB131 medium with 2% BSA for maturation of insulin-positive endocrinecells into SC-β cells can be comprise no small molecule factors asdescribed herein. In some case, the MCDB131 medium with 2% BSA formaturation of insulin-positive endocrine cells into SC-β cells cancomprise no serum (e.g., no FBS).

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) aTGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator,iii) at least one SHH pathway inhibitor, iv) a RA signaling pathwayactivator, v) a γ-secretase inhibitor, optionally vi) at least onegrowth factor from the epidermal growth factor (EGF) family, andoptionally vii) a BMP signaling pathway inhibitor, every other day for aperiod of between five and seven days; and f) differentiating at leastsome of the Pdx1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells by a process of culturing the Pdx1-positive,NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g.,NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, orDMEM/F12 medium) without exogenous differentiation factors, every otherday for a period of between 7 and 14 days to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some cases, the GSISresponse resembles the GSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor, v) a PKCactivator, vi) a ROCK inhibitor, and vii) a growth factor from TGFβsuperfamily, for a period of 2 days; d) differentiating at least some ofthe Pdx1-positive pancreatic progenitor cells into Pdx1-positive,NKX6.1-positive pancreatic progenitor cells by a process of contactingthe Pdx1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with i) at least one growth factor from the FGFfamily, ii) at least one SHH pathway inhibitor, and optionally iii) a RAsignaling pathway activator, and optionally iv) ROCK inhibitor and v) atleast one factor from TGFβ superfamily, every other day for a period of5 days, wherein the NKX6.1-positive pancreatic progenitor cellsexpresses Pdx1 and NKX6.1; e) differentiating at least some of thePdx1-positive, NKX6.1-positive pancreatic progenitor cells intoPdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a THsignaling pathway activator, iii) at least one SHH pathway inhibitor,iv) a RA signaling pathway activator, v) a γ-secretase inhibitor,optionally vi) at least one growth factor from the epidermal growthfactor (EGF) family, and optionally vii) a BMP signaling pathwayinhibitor, every other day for a period of between five and seven days;and f) differentiating at least some of the Pdx1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by aprocess of culturing the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells in a medium (e.g., NS-GFs medium, MCDBmedium supplemented with BSA, MCDB131 medium, or DMEM/F12 medium)without exogenous differentiation factors, every other day for a periodof between 7 and 14 days to induce the in vitro maturation of at leastsome of the Pdx1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells, wherein the SC-β cells exhibit a GSIS response invitro and/or in vivo. In some cases, the GSIS response resembles theGSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) aTGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator,iii) at least one SHH pathway inhibitor, iv) a RA signaling pathwayactivator, v) a γ-secretase inhibitor, and optionally vi) at least onegrowth factor from the epidermal growth factor (EGF) family, every otherday for a period of between five and seven days; and f) differentiatingat least some of the Pdx1-positive, NKX6.1-positive, insulin-positiveendocrine cells into SC-β cells by a process of culturing thePdx1-positive, NKX6.1-positive, insulin-positive endocrine cells in amedium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131medium, or DMEM/F12 medium) without exogenous differentiation factors,every other day for a period of between 7 and 14 days to induce the invitro maturation of at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some cases, the GSISresponse resembles the GSIS response of an endogenous mature β cells.

The medium used to culture the cells dissociated from the first cellcluster can be xeno-free. A xeno-free medium for culturing cells and/orcell clusters of originated from an animal can have no product fromother animals. In some cases, a xeno-free medium for culturing humancells and/or cell clusters can have no products from any non-humananimals. For example, a xeno-free medium for culturing human cellsand/or cell clusters can comprise human platelet lysate (PLT) instead offetal bovine serum (FBS). For example, a medium can comprise from about1% to about 20%, from about 5% to about 15%, from about 8% to about 12%,from about 9 to about 11% serum. In some cases, medium can compriseabout 10% of serum. In some cases, the medium can be free of smallmolecules and/or FBS. For example, a medium can comprise MCDB131 basalmedium supplemented with 2% BSA. In some cases, the medium isserum-free. In some examples, a medium can comprise no exogenous smallmolecules or signaling pathway agonists or antagonists, such as, growthfactor from fibroblast growth factor family (FGF, such as FGF2, FGF8B,FGF 10, or FGF21), Sonic Hedgehog Antagonist (such as Sant1, Sant2,Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol,cyclopamine, or derivatives thereof), Retinoic Acid Signaling agonist(e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961,AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314),inhibitor of Rho-associated, coiled-coil containing protein kinase(ROCK) (e.g., Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152),activator of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate(PDBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, orderivatives thereof), antagonist of TGF 13 super family (e.g., Alk5inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388,LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, orderivatives thereof), inhibitor of Bone Morphogenetic Protein (BMP) type1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormonesignaling pathway activator (e.g., T3 or derivatives thereof),gamma-secretase inhibitor (e.g., XXI, DAPT, or derivatives thereof),activator of TGF-β signaling pathway (e.g., WNT3a or Activin A) growthfactor from epidermal growth factor (EGF) family (e.g., betacellulin orEGF), broad kinase (e.g., staurosporine or derivatives thereof),non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine,vitamin D, vitamin C, vitamin A, or derivatives thereof), or otheradditions like N-acetyl cysteine, zinc sulfate, or heparin. In somecases, the reaggregation medium can comprise no exogenous extracellularmatrix molecule. In some cases, the reaggregation medium does notcomprise Matrigel™. In some cases, the reaggregation medium does notcomprise other extracellular matrix molecules or materials, such as,collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin,fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixturesthereof, for example, or lysed cell membrane preparations.

A person of ordinary skill in the art will appreciate that that theconcentration of serum albumin supplemented into the medium may vary.For example, a medium (e.g., MCDB131) can comprise about 0.01%, 0.05%,0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about15% BSA. In other cases, a medium can comprise about 0.01%, 0.05%, 0.1%,1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA.The medium used (e.g., MCDB131 medium) can contain components not foundin traditional basal media, such as trace elements, putrescine, adenine,thymidine, and higher levels of some amino acids and vitamins. Theseadditions can allow the medium to be supplemented with very low levelsof serum or defined components. The medium can be free of proteinsand/or growth factors, and may be supplemented with EGF, hydrocortisone,and/or glutamine. The medium can comprise one or more extracellularmatrix molecules (e.g., extracellular proteins). Non-limiting exemplaryextracellular matrix molecules used in the medium can include collagen,placental matrix, fibronectin, laminin, merosin, tenascin, heparin,heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan,biglycan, thrombospondin, vitronectin, and decorin. In some cases, themedium comprises laminin, such as LN-332. In some cases, the mediumcomprises heparin.

The medium can be changed periodically in the culture, e.g., to provideoptimal environment for the cells in the medium. When culturing thecells dissociated from the first cell cluster for re-aggregation, themedium can be changed at least or about every 4 hours, 12 hours, 24hours, 48 hours, 3 days or 4 days. For example, the medium can bechanged about every 48 hours.

In some cases, cells can be cultured under dynamic conditions (e.g.,under conditions in which the cells are subject to constant movement orstirring while in the suspension culture). For dynamic culturing ofcells, the cells can be cultured in a container (e.g., an non-adhesivecontainer such as a spinner flask (e.g., of 200 ml to 3000 ml, forexample 250 ml; of 100 ml; or in 125 ml Erlenmeyer), which can beconnected to a control unit and thus present a controlled culturingsystem. In some cases, cells can be cultured under non-dynamicconditions (e.g., a static culture) while preserving their proliferativecapacity. For non-dynamic culturing of cells, the cells can be culturedin an adherent culture vessel. An adhesive culture vessel can be coatedwith any of substrates for cell adhesion such as extracellular matrix(ECM) to improve the adhesiveness of the vessel surface to the cells.The substrate for cell adhesion can be any material intended to attachstem cells or feeder cells (if used). The substrate for cell adhesionincludes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin,laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin andmixtures thereof, for example, Matrigel™, and lysed cell membranepreparations.

Medium in a dynamic cell culture vessel (e.g., a spinner flask) can bestirred (e.g., by a stirrer). The spinning speed can correlate with thesize of the re-aggregated second cell cluster. The spinning speed can becontrolled so that the size of the second cell cluster can be similar toan endogenous pancreatic islet. In some cases, the spinning speed iscontrolled so that the size of the second cell cluster can be from about75 μm to about 250 μm. The spinning speed of a dynamic cell culturevessel (e.g., a spinner flask) can be about 20 rounds per minute (rpm)to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some cases,the spinning speed can be about 50 rpm.

Stage 6 cells as provided herein may or may not be subject to thedissociation and reaggregation process as described herein. In somecases, the cell cluster comprising the insulin-positive endocrine cellscan be reaggregated. The reaggregation of the cell cluster can enrichthe insulin-positive endocrine cells. In some cases, theinsulin-positive endocrine cells in the cell cluster can be furthermatured into pancreatic β cells. For example, after reaggregation, thesecond cell cluster can exhibit in vitro GSIS, resembling nativepancreatic islet. For example, after reaggregation, the second cellcluster can comprise non-native pancreatic β cell that exhibits in vitroGSIS. In some embodiments, the reaggregation process can be performedaccording to the disclosure of PCT application PCT/US2018/043179, whichis incorporated herein by reference in its entirety.

In some embodiments, the present disclosure relates to cryopreservationof the non-native pancreatic β cells or precursors thereof obtainedusing the methods provided herein. In some embodiments, the cellpopulation comprising non-native pancreatic β cells can be stored viacryopreservation. For instances, the cell population comprisingnon-native β cells, e.g., Stage 6 cells in some cases, can bedissociated into cell suspension, e.g., single cell suspension, and thecell suspension can be cryopreserved, e.g., frozen in a cryopreservationsolution. The dissociation of the cells can be conducted by any of thetechnique provided herein, for example, by enzymatic treatment. Thecells can be frozen at a temperature of at highest −20° C., at highest−30° C., at highest −40° C., at highest −50° C., at highest −60° C., athighest −70° C., at highest −80° C., at highest −90° C., at highest−100° C., at highest −110° C., at highest −120° C., at highest −130° C.,at highest −140° C., at highest −150° C., at highest −160° C., athighest −170° C., at highest −180° C., at highest −190° C., or athighest −200° C. In some cases, the cells are frozen at a temperature ofabout −80° C. In some cases, the cells are frozen at a temperature ofabout −195° C. Any cooling methods can be used for providing the lowtemperature needed for cryopreservation, such as, but not limited to,electric freezer, solid carbon dioxide, and liquid nitrogen. In somecases, any cryopreservation solution available to one skilled in the artcan be used for incubating the cells for storage at low temperature,including both custom made and commercial solutions. For example, asolution containing a cryoprotectant can be used. The cryoprotectant canbe an agent that is configured to protect the cell from freezing damage.For instance, a cryoprotectant can be a substance that can lower theglass transition temperature of the cryopreservation solution. Exemplarycryoprotectants that can be used include DMSO (dimethyl sulfoxide),glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran(e.g., dextran-40), and trehalose. Additional agents can be added in tothe cryopreservation solution for other effects. In some cases,commercially available cryopreservation solutions can be used in themethod provided herein, for instance, FrostaLife™, pZerve™, PrimeXV®,Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLBANKER®,CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, andCryoDefend® Stem Cells Media.

V. Differentiation Factors

Aspects of the disclosure relate to contacting progenitor cells (e.g.,stem cells, e.g., iPS cells, definitive endoderm cells, primitive guttube cells, Pdx1-positive pancreatic progenitor cells, NKX6.1-positivepancreatic progenitor cells, insulin-positive endocrine cells) with βcell differentiation factors, for example, to induce the maturation ofthe insulin-positive endocrine cells or differentiation of otherprogenitor cells into SC-β cells (e.g., mature pancreatic β cells). Insome embodiments, the differentiation factor can induce thedifferentiation of pluripotent cells (e.g., iPSCs or hESCs) intodefinitive endoderm cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of definitive endoderm cells into primitive gut tubecells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof primitive gut tube cells into Pdx1-positive pancreatic progenitorcells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof Pdx1-positive pancreatic progenitor cells into NKX6-1-positivepancreatic progenitor cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of NKX6-1-positive pancreatic progenitor cells intoinsulin-positive endocrine cells, e.g., in accordance with a methoddescribed herein. In some embodiments, the differentiation factor caninduce the maturation of insulin-positive endocrine cells into SC-βcells, e.g., in accordance with a method described herein.

At least one differentiation factor described herein can be used alone,or in combination with other differentiation actors, to generate SC-βcells according to the methods as disclosed herein. In some embodiments,at least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tendifferentiation factors described herein are used in the methods ofgenerating SC-β cells.

Transforming Growth Factor-β (TGF-β) Superfamily

Aspects of the disclosure relate to the use of growth factors from thetransforming growth factor-β (TGF-β) superfamily as differentiationfactors. The “TGF-β superfamily” means proteins having structural andfunctional characteristics of known TGFβ family members. The TGFβ familyof proteins can include the TGFβ series of proteins, the Inhibins(including Inhibin A and Inhibin B), the Activins (including Activin A,Activin B, and Activin AB), MIS (Müllerian inhibiting substance), BMP(bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF(monoclonal nonspecific suppressor factor), and others. Activity of thisfamily of proteins can be based on specific binding to certain receptorson various cell types. Members of this family can share regions ofsequence identity, particularly at the C-terminus, that correlate totheir function. The TGFβ family can include more than one hundreddistinct proteins, all sharing at least one region of amino acidsequence identity. Members of the family that can be used in the methoddisclosed herein can include, but are not limited to, the followingproteins, as identified by their GenBank accession numbers: P07995,P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529,P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959, O08717,P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643,P49001, P21274, O46564, O19006, P22004, P20722, Q04906, Q07104, P30886,P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885,P34820, Q29607, P12644, Q90752, O46576, P27539, P48969, Q26974, P07713,P91706, P91699, P27091, O42222, Q24735, P20863, O18828, P55106, Q9PTQ2,O14793, O08689, O42221, O18830, O18831, O18836, O35312, O42220, P43026,P43027, P43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, P43028, Q7Z4P5,P50414, P17246, P54831, P04202, P01137, P09533, P18341, O19011, Q9Z1Y6,P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737,AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1 S4Y, NP-001009856.1,NP-1-032406.1, NP-999193.1, XP-519063.1, AAG17260.1, CAA40806.1,NP-1-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1,AAC59738.1, CAI46003.1, B40905, AAQ55811.1, AAK40342.1, XP-540364.1,P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504,BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG05339.1, BAB17601.1,CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1,AAA59169.1, XP-541000.1, NP-990537.1, NP-1-002184.1, AAC14187.1,AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C,AAH30029.1, CAA49326.1, XP-344131.1, AA-148845.1, XP-1-148966.3, 148235,B41398, AAH77857.1, AAB26863.1, 1706327A, BAA83804.1, NP-571143.1,CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506,CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1, BAC81672.1, BAA12694.1,BAA08494.1, B36192, C36192, BAB16971.1, NP-034695.1, AAA49160.1,CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1,XP-522443.1, AAM27448.1, XP-538247.1, AAD30133. I, AAC36741.1,AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1, AAC32311.1,NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-571023.1,CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1,AAH73508.1, AAD02201.1, NP-999793.1, NP-990542.1, AAF19841.1,AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1, EAL41229.1,AAT07302.1, CAI19472.1, NP-031582.1, AAA40548.1, XP-535880.1,NP-1-037239.1, AAT72007.1, XP-418956.1, CAA41634.1, BAC30864.1,CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1,NP-990479.1, NP-999820.1, AAB27335.1, 545355, CAB82007.1, XP-534351.1,NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1,BAA89012.1, IES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1,AAQ89234.1, AAM27000.1, AAH30959.1, CAG01491.1, NP-571435.1, 1REU,AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1,CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.1,AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1,XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1,CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1,AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AATI2416.1,NP-571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1,BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1,BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1,AAB94786.1, NP-001483.2, XP-528195.1, NP-571417.1, NP-001001557. I,AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2,P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1,NP-1-038554.1, CAG04089.1, CAD60936.2, NP-031584.1, B55452, AAC60285.1,BAA06410.1, AAH52846.1, NP-031580.1, NP-1-036959.1, CAA45836.1,CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1,AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1,AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1,BAC02895.1, AAWS 1451.1, AAF82188.1, XP-544189.1, NP-990568.1,BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1,AAT99303.1, AAD28038.1, AAH52168.1, NP-001004122.1, CAA72733.1,NP-032133.2, XP-394252.1, XP-224733.2, JH10801, AAP97721.1, NP-989669.1,543296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032135.1,AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1,AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1,CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1,CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1,AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1,AAW47740.1, AAW29442.1, NP-722813.1, AAR08901.1, AAO 15420.2,CAC59700.1, AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1,AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1,NP-005802.1, XP-343149.1, AW34055.1, XP-538221.1, AAR27580.1,XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1 W4, NP-1-031585.2,NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1,CAE46407.1, XP-1-417667.1, BAC53989.1, BAB19659.1, AAM46922.1,AAA81169.1, AAK28707.1, AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1,BAD16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1,AAK08152.2, 2009388A, AAR12161.1, CAGO1961.1, CAB63656.1, CAD67714.1,CAF94162.1, NP-477340.1, EAL24792.1, NP-1-001009428.1, AAB86686.1,AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1,Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1,XP-525772.1, NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1,AAH74757.2, NP-034964.1, NP-038639.1, O42221, AAF02773.1, NP-062024.1,AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1,AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1,BAB16046.1, AAN63522.1, NP-571041.1, AAB04986.2, AAC26791.1, AAB95254.1,BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1,XP-1-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1,AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1,AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2,NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997. I, AAN03842.1,NP-571951.2, CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1,NP-624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2,NP-001192.1, XP-544948.1, AAQ18013.1, AAV38739.1, NP-851298.1,CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1,XP-1-421648.1, CAB63704.1, NP-037306.1, A55706, AAF02780.1, CAG09623.1,NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1,AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP-1-001009400.1,AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1,AAA31526.1.

The growth factor from the TGF-β superfamily in the methods andcompositions provided herein can be naturally obtained or recombinant.In some embodiments, the growth factor from the TGF-β superfamilycomprises Activin A. The term “Activin A” can include fragments andderivatives of Activin A. The sequence of an exemplary Activin A isdisclosed as SEQ ID NO: 1 in U.S. Pub. No. 2009/0155218 (the '218publication). Other non-limiting examples of Activin A are provided inSEQ ID NO: 2-16 of the '218 publication, and non-limiting examples ofnucleic acids encoding Activin A are provided in SEQ ID NO:33-34 of the'218 publication. In some embodiments, the growth factor from the TGF-βsuperfamily can comprise a polypeptide having an amino acid sequence atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%, or greateridentical to SEQ ID NO: 1 of the '218 publication.

In some embodiments, the growth factor from the TGF-β superfamilycomprises growth differentiation factor 8 (GDF8). The term “GDF8” caninclude fragments and derivatives of GDF8. The sequences of GDF8polypeptides are available to the skilled artisan. In some embodiments,the growth factor from the TGF-β superfamily comprises a polypeptidehaving an amino acid sequence at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%, or greater identical to the human GDF8 polypeptidesequence (GenBank Accession EAX10880).

In some embodiments, the growth factor from the TGF-β superfamilycomprises a growth factor that is closely related to GDF8, e.g., growthdifferentiation factor 11 (GDF11). In some embodiments, the growthfactor from the TGF-β superfamily comprises a polypeptide having anamino acid sequence at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%, or greater identical to the human GDF11 polypeptide sequence(GenBank Accession AAF21630).

In some embodiments, the growth factor from the TGF-β superfamily can bereplaced with an agent mimics the at least one growth factor from theTGF-β superfamily. Exemplary agents that mimic the at least one growthfactor from the TGF-β superfamily, include, without limitation, IDE1 andIDE2.

Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of BMP signaling pathwayinhibitors as β cell differentiation factors. The BMP signaling familyis a diverse subset of the TGF-β superfamily (Sebald et al. Biol. Chem.385:697-710, 2004). Over twenty known BMP ligands are recognized bythree distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least threetype I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitateassembly of receptor heteromers, allowing the constitutively-active typeII receptor serine/threonine kinases to phosphorylate type I receptorserine/threonine kinases. Activated type I receptors phosphorylateBMP-responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitatenuclear translocation in complex with SMAD4, a co-SMAD that alsofacilitates TGF signaling. In addition, BMP signals can activateintracellular effectors such as MAPK p38 in a SMAD-independent manner(Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists suchas noggin, chordin, gremlin, and follistatin limit BMP signaling byligand sequestration.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises DMH-1, or a derivative,analogue, or variant thereof. In some embodiments, the BMP signalingpathway inhibitor in the methods and composition provided hereincomprises the following compound or a derivative, analogue, or variantof the following compound:

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises LDN193189 (also known asLDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC Name:4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises the following compound or aderivative, analogue, or variant of the following compound:

In some cases, DMH-1 can be more selective as compared to LDN193189. Insome embodiments of the present disclosure, DMH-1 can be particularlyuseful for the methods provided herein. In some embodiments, the methodsand compositions provided herein exclude use of LDN193189. In someembodiments, the methods and compositions provided herein exclude use ofLDN193189, or a derivative, analogue, or variant thereof for generatingPdx1-positive pancreatic progenitor cells from primitive gut tube cells.In some embodiments, the methods and compositions provided herein relateto use of DMH-1, or a derivative, analogue, or variant thereof forgenerating Pdx1-positive pancreatic progenitor cells from primitive guttube cells.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprise an analog or derivative ofLDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug ofLDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189comprises LDN193189 hydrochloride.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises a compound of Formula I fromU.S. Patent Publication No. 2011/0053930.

TGF-β Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of TGF-β signaling pathwayinhibitors as β cell differentiation factors.

In some embodiments, the TGF-β signaling pathway comprises TGF-βreceptor type I kinase (TGF-β RI) signaling. In some embodiments, theTGF-β signaling pathway inhibitor comprises ALK5 inhibitor II (CAS446859-33-2, an ATP-competitive inhibitor of TGF-B RI kinase, also knownas RepSox, IUPAC Name:2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine. In someembodiments, the TGF-β signaling pathway inhibitor is an analog orderivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II (alsonamed “ALK5i”) is a compound of Formula I as described in U.S. PatentPublication No. 2012/0021519, incorporated by reference herein in itsentirety.

In some embodiments, the TGF-β signaling pathway inhibitor in themethods and compositions provided herein is a TGF-β receptor inhibitordescribed in U.S. Patent Publication No. 2010/0267731. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein comprises an ALK5 inhibitor described inU.S. Patent Publication Nos. 2009/0186076 and 2007/0142376. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is not A 83-01. In some embodiments, the compositions and methodsdescribed herein exclude A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is SB 431542. In some embodiments, the TGF-β signaling pathwayinhibitor is not SB 431542. In some embodiments, the compositions andmethods described herein exclude SB 431542. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is D 4476. In some embodiments, the TGF-β signalingpathway inhibitor is not D 4476. In some embodiments, the compositionsand methods described herein exclude D 4476. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is GW 788388. In some embodiments, the TGF-β signalingpathway inhibitor is not GW 788388. In some embodiments, thecompositions and methods described herein exclude GW 788388. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 364947. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 364947. In some embodiments,the compositions and methods described herein exclude LY 364947. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 580276. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 580276. In some embodiments,the compositions and methods described herein exclude LY 580276. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 525334. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 525334. In some embodiments,the compositions and methods described herein exclude SB 525334. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 505124. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 505124. In some embodiments,the compositions and methods described herein exclude SB 505124. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SD 208. In some embodiments, the TGF-βsignaling pathway inhibitor is not SD 208. In some embodiments, thecompositions and methods described herein exclude SD 208. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 6604. In some embodiments, the TGF-βsignaling pathway inhibitor is not GW 6604. In some embodiments, thecompositions and methods described herein exclude GW 6604. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 788388. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is not GW 788388. In some embodiments, the compositionsand methods described herein exclude GW 788388.

From the collection of compounds described above, the following can beobtained from various sources: LY-364947, SB-525334, SD-208, andSB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo.,63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals,Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604available from GlaxoSmithKline, 980 Great West Road, Brentford,Middlesex, TW8 9GS, United Kingdom; LY580276 available from LillyResearch, Indianapolis, Ind. 46285; and SM16 available from Biogen Idec,P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C.,27709-4627.

WNT Signaling Pathway

Aspects of the disclosure relate to the use of activators of the WNTsignaling pathway as β cell differentiation factors.

In some embodiments, the WNT signaling pathway activator in the methodsand compositions provided herein comprises CHIR99021. In someembodiments, the WNT signaling pathway activator in the methods andcompositions provided herein comprises a derivative of CHIR99021, e.g.,a salt of CHIR99021, e.g., trihydrochloride, a hydrochloride salt ofCHIR99021. In some embodiments, the WNT signaling pathway activator inthe methods and compositions provided herein comprises Wnt3a recombinantprotein. In some embodiments, the WNT signaling pathway activator in themethods and compositions provided herein comprises a glycogen synthasekinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, withoutlimitation, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide,10Z-Hymenialdisine, Indirubin-3′oxime, kenpaullone, L803, L803-mts,lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002,TCS 21311, TWS 119, and analogs or derivatives of any of these. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a WNT signaling pathway activator.

Fibroblast Growth Factor (FGF) Family

Aspects of the disclosure relate to the use of growth factors from theFGF family as β cell differentiation factors.

In some embodiments, the growth factor from the FGF family in themethods and compositions provided herein comprises keratinocyte growthfactor (KGF). The polypeptide sequences of KGF are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human KGF polypeptide sequence (GenBank Accession AAB21431).

In some embodiments, the growth factor from the FGF family in themethods and composition provided herein comprises FGF2. The polypeptidesequences of FGF2 are available to the skilled artisan. In someembodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF2polypeptide sequence (GenBank Accession NP001997).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF8B. Thepolypeptide sequences of FGF8B are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF8Bpolypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF10. Thepolypeptide sequences of FGF10 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF10polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF21. Thepolypeptide sequences of FGF21 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF21polypeptide sequence (GenBank Accession AAQ89444.1).

Sonic Hedgehog (SHH) Signaling Pathway

Aspects of the disclosure relate to the use of SHH signaling pathwayinhibitors as β cell differentiation factors.

In some embodiments, the SHH signaling pathway inhibitor in the methodsand composition provided herein comprises Sant1. In some embodiments,the SHH signaling pathway inhibitor in the methods and compositionprovided herein comprises SANT2. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises SANT3. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesSANT4. In some embodiments, the SHH signaling pathway inhibitorcomprises Cur61414. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesforskolin. In some embodiments, the SHH signaling pathway inhibitor inthe methods and composition provided herein comprises tomatidine. Insome embodiments, the SHH signaling pathway inhibitor in the methods andcomposition provided herein comprises AY9944. In some embodiments, theSHH signaling pathway inhibitor in the methods and composition providedherein comprises triparanol. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises compound A or compound B (as disclosed in U.S. Pub. No.2004/0060568). In some embodiments, the SHH signaling pathway inhibitorin the methods and composition provided herein comprises a steroidalalkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or aderivative thereof) as disclosed in U.S. Pub. No. 2006/0276391. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a SHH signaling pathway inhibitor.

Retinoic Acid Signaling Pathway

Aspects of the disclosure relate to the use of modulators of retinoicacid signaling as β cell differentiation factors.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an activator ofretinoic acid signaling. In some embodiments, the RA signaling pathwayactivator in the methods and composition provided herein comprisesretinoic acid. In some embodiments, the RA signaling pathway activatorin the methods and composition provided herein comprises a retinoic acidreceptor agonist. Exemplary retinoic acid receptor agonists in themethods and composition provided herein include, without limitation, CD1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80,BMS 753, tazarotene, adapalene, and CD 2314.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an inhibitor ofretinoic acid signaling. In some embodiments, the retinoic acidsignaling pathway inhibitor comprises DEAB (IUPAC Name:2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In someembodiments, the retinoic acid signaling pathway inhibitor comprises ananalog or derivative of DEAB.

In some embodiments, the retinoic acid signaling pathway inhibitor inthe methods and composition provided herein comprises a retinoic acidreceptor antagonist. In some embodiments, the retinoic acid receptorantagonist in the methods and composition provided herein comprises(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoicacid,(E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoicacid,(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]-benzoicacid, and(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoicacid. In some embodiments, the retinoic acid receptor antagonistcomprises BMS 195614 (CAS #253310-42-8), ER 50891 (CAS #187400-85-7),BMS 493 (CAS #170355-78-9), CD 2665 (CAS #170355-78-9), LE 135 (CAS#155877-83-1), BMS 453 (CAS #166977-43-1), or MM 11253 (CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosedherein exclude a modulator of retinoic acid signaling. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway activator. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway inhibitor.

Protein Kinase C

Aspects of the disclosure relate to the use of protein kinase Cactivators as β cell differentiation factors. Protein kinase C is one ofthe largest families of protein kinase enzymes and is composed of avariety of isoforms. Conventional isoforms include a, βI, γ; novelisoforms include δ, ε, η, Θ; and atypical isoforms include ξ and ι/λ.PKC enzymes are primarily cytosolic but translocate to the membrane whenactivated. In the cytoplasm, PKC is phosphorylated by other kinases orautophosphorylates. In order to be activated, some PKC isoforms (e.g.,PKC-ε) require a molecule to bind to the diacylglycerol (“DAG”) bindingsite or the phosphatidylserine (“PS”) binding site. Others are able tobe activated without any secondary binding messengers at all. PKCactivators that bind to the DAG site include, but are not limited to,bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin.PKC activators that bind to the PS site include, but are not limited to,polyunsaturated fatty acids and their derivatives. It is contemplatedthat any protein kinase C activator that is capable, either alone or incombination with one or more other β cell differentiation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the PKC activator in the methods and compositionprovided herein comprises PdbU. In some embodiments, the PKC activatorin the methods and composition provided herein comprises TPB. In someembodiments, the PKC activator in the methods and composition providedherein comprises cyclopropanated polyunsaturated fatty acids,cyclopropanated monounsaturated fatty acids, cyclopropanatedpolyunsaturated fatty alcohols, cyclopropanated monounsaturated fattyalcohols, cyclopropanated polyunsaturated fatty acid esters,cyclopropanated monounsaturated fatty acid esters, cyclopropanatedpolyunsaturated fatty acid sulfates, cyclopropanated monounsaturatedfatty acid sulfates, cyclopropanated polyunsaturated fatty acidphosphates, cyclopropanated monounsaturated fatty acid phosphates,macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V,gnidimacrin, iripallidal, ingenol, napthalenesulfonamides,diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18),insulin growth factor, hormones, and growth factor activators, asdescribed in WIPO Pub. No. WO/2013/071282. In some embodiments, thebryostain comprises bryostatin-1, bryostatin-2, bryostatin-3,bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8,bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12,bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16,bryostatin-17, or bryostatin-18. In certain embodiments, the methods,compositions, and kits disclosed herein exclude a protein kinase Cactivator.

γ-Secretase Inhibitors

Aspects of the disclosure relate to the use of γ-secretase inhibitors asβ cell differentiation factors.

In some embodiments, the γ-secretase inhibitor in the methods andcomposition provided herein comprises XXI. In some embodiments, theγ-secretase inhibitor in the methods and composition provided hereincomprises DAPT. Additional exemplary γ-secretase inhibitors in themethods and composition provided herein include, without limitation, theγ-secretase inhibitors described in U.S. Pat. Nos. 7,049,296, 8,481,499,8,501,813, and WIPO Pub. No. WO/2013/052700. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a γ-secretaseinhibitor.

Thyroid Hormone Signaling Pathway Activators

Aspects of the disclosure relate to the use of thyroid hormone signalingpathway activators as β cell differentiation factors.

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises triiodothyronine(T3). In some embodiments, the thyroid hormone signaling pathwayactivator in the methods and composition provided herein comprises ananalog or derivative of T3. Exemplary analogs of T3 in the methods andcomposition provided herein include, but are not limited to, selectiveand non-selective thyromimetics, TRβ selective agonist-GC-1,GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid(DITPA); the selective TR-β agonist GC-1; 3-Iodothyronamine (T(1)AM) and3,3′,5-triiodothyroacetic acid (Triac) (bioactive metabolites of thehormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901;DIBIT; 3′-AC-T2; tetraiodothyroacetic acid (Tetrac) andtriiodothyroacetic acid (Triac) (via oxidative deamination anddecarboxylation of thyroxine [T4] and triiodothyronine [T3] alaninechain), 3,3′,5′-triiodothyronine (rT3) (via T4 and T3 deiodination),3,3′-diiodothyronine (3,3′-T2) and 3,5-diiodothyronine (T2) (via T4, T3,and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine(TOAM) (via T4 and T3 deiodination and amino acid decarboxylation), aswell as for TH structural analogs, such as 3,5,3′-triiodothyropropionicacid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L-940901),N-[3,5-dimethyl-4-(4′-hydroxy-3′-isopropylphenoxy)-phenyl]-oxamic acid(CGS 23425),3,5-dimethyl-4-[(4′-hydroxy-3′-isopropylbenzyl)-phenoxy]acetic acid(GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid(KB-141), and 3,5-diiodothyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises a prodrug orprohormone of T3, such as T4 thyroid hormone (e.g., thyroxine orL-3,5,3′,5′-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein is an iodothyroninecomposition described in U.S. Pat. No. 7,163,918.

Epidermal Growth Factor (EGF) Family

Aspects of the disclosure relate to the use of growth factors from theEGF family as β cell differentiation factors.

In some embodiments, the at least one growth factor from the EGF familyin the methods and composition provided herein comprises betacellulin.In some embodiments, at least one growth factor from the EGF family inthe methods and composition provided herein comprises EGF. Epidermalgrowth factor (EGF) is a 53 amino acid cytokine which is proteolyticallycleaved from a large integral membrane protein precursor. In someembodiments, the growth factor from the EGF family in the methods andcomposition provided herein comprises a variant EGF polypeptide, forexample an isolated epidermal growth factor polypeptide having at least90% amino acid identity to the human wild-type EGF polypeptide sequence,as disclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growthfactor from the EGF family in the methods and composition providedherein comprises an engineered EGF mutant that binds to and agonizes theEGF receptor, as is disclosed in U.S. Pat. No. 8,247,531. In someembodiments, the at least one growth factor from the EGF family in themethods and composition provided herein is replaced with an agent thatactivates a signaling pathway in the EGF family. In some embodiments,the growth factor from the EGF family in the methods and compositionprovided herein comprises a compound that mimics EGF. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a growth factor from the EGF family.

Protein Kinase Inhibitors

Aspects of the disclosure relate to the use of protein kinase inhibitorsas β cell differentiation factors.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein comprises staurosporine. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein comprises an analog of staurosporine. Exemplary analogsof staurosporine in the methods and composition provided herein include,without limitation, Ro-31-8220, a bisindolylmaleimide (Bis) compound,10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine,a staralog (see, e.g., Lopez et al., “Staurosporine-derived inhibitorsbroaden the scope of analog-sensitive kinase technology”, J. Am. Chem.Soc. 2013; 135(48):18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein is an inhibitor of PKCβ. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein is an inhibitor of PKCβ with the following structure ora derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKCβ is a GSK-2 compound with thefollowing structure or a derivative, analogue or variant of the compoundas follows:

In some embodiments, the inhibitor of PKC in the methods and compositionprovided herein is a bisindolylmaleimide. Exemplary bisindolylmaleimidesinclude, without limitation, bisindolylmaleimide I, bisindolylmaleimideII, bisindolylmaleimide Ill, hydrochloride, or a derivative, analogue orvariant thereof.

In some embodiments, the PKC inhibitor in the methods and compositionprovided herein is a pseudohypericin, or a derivative, analogue, orvariant thereof. In some embodiments, the PKC inhibitor in the methodsand composition provided herein is indorublin-3-monoximc, 5-Iodo or aderivative, analogue or variant thereof. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a proteinkinase inhibitor.

VI. Pharmaceutical Compositions

In some cases, the present disclosure provides pharmaceuticalcompositions that can utilize non-native pancreatic β cell (beta cells)populations and cell components and products in various methods fortreatment of a disease (e.g., diabetes). Certain cases encompasspharmaceutical compositions comprising live cells (e.g., non-nativepancreatic β cells alone or admixed with other cell types). Other casesencompass pharmaceutical compositions comprising non-native pancreatic βcell components (e.g., cell lysates, soluble cell fractions, conditionedmedium, ECM, or components of any of the foregoing) or products (e.g.,trophic and other biological factors produced by non-native pancreatic βcells or through genetic modification, conditioned medium fromnon-native pancreatic β cell culture). In either case, thepharmaceutical composition may further comprise other active agents,such as anti-inflammatory agents, exogenous small molecule agonists,exogenous small molecule antagonists, anti-apoptotic agents,antioxidants, and/or growth factors known to a person having skill inthe art.

Pharmaceutical compositions of the present disclosure can comprisenon-native pancreatic β cell, or components or products thereof,formulated with a pharmaceutically acceptable carrier (e.g. a medium oran excipient). The term pharmaceutically acceptable carrier (or medium),which may be used interchangeably with the term biologically compatiblecarrier or medium, can refer to reagents, cells, compounds, materials,compositions, and/or dosage forms that are not only compatible with thecells and other agents to be administered therapeutically, but also aresuitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or othercomplication. Suitable pharmaceutically acceptable carriers can includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring. Pharmaceutical compositions comprising cellular components orproducts, but not live cells, can be formulated as liquids.Pharmaceutical compositions comprising living non-native pancreatic βcells can be formulated as liquids, semisolids (e.g., gels, gelcapsules, or liposomes) or solids (e.g., matrices, scaffolds and thelike).

As used here, the term “pharmaceutically acceptable” can refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” can referto a pharmaceutically-acceptable material, composition or vehicle, suchas a liquid or solid filler, diluent, excipient, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient,” “carrier,” “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein in respectto a population of cells means that amount of relevant cells in apopulation of cells, e.g., SC-β cells or mature pancreatic β cells, orcomposition comprising SC-β cells of the present disclosure which iseffective for producing some desired therapeutic effect in at least asub-population of cells in an animal at a reasonable benefit/risk ratioapplicable to any medical treatment. For example, an amount of apopulation of SC-β cells administered to a subject that is sufficient toproduce a statistically significant, measurable change in at least onesymptom of Type 1, Type 1.5 or Type 2 diabetes, such as glycosylatedhemoglobin level, fasting blood glucose level, hypoinsulinemia, etc.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

Pharmaceutical compositions can comprise auxiliary components as wouldbe familiar to a person having skill in the art. For example, they cancontain antioxidants in ranges that vary depending on the kind ofantioxidant used. Reasonable ranges for commonly used antioxidants areabout 0.01% to about 0.15% weight by volume of EDTA, about 0.01% toabout 2.0% weight volume of sodium sulfite, and about 0.01% to about2.0% weight by volume of sodium metabisulfite. One skilled in the artmay use a concentration of about 0.1% weight by volume for each of theabove. Other representative compounds include mercaptopropionyl glycine,N-acetyl cysteine, β-mercaptoethylamine, glutathione and similarspecies, although other anti-oxidant agents suitable for renaladministration, e.g. ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent can be used to maintain the pH of formulations in therange of about 4.0 to about 8.0; so as to minimize irritation in thetarget tissue. For direct intraperitoneal injection, formulations shouldbe at pH 7.2 to 7.5, preferably at pH 7.35-7.45. The compositions mayalso include tonicity agents suitable for administration to the kidney.Among those suitable is sodium chloride to make formulationsapproximately isotonic with blood.

In certain cases, pharmaceutical compositions are formulated withviscosity enhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Thepharmaceutical compositions may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives mayalso be included, e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methylor propylparabens.

Pharmaceutical compositions comprising cells, cell components or cellproducts may be delivered to the kidney of a patient in one or more ofseveral methods of delivery known in the art. In some cases, thecompositions are delivered to the kidney (e.g., on the renal capsuleand/or underneath the renal capsule). In another embodiment, thecompositions may be delivered to various locations within the kidney viaperiodic intraperitoneal or intrarenal injection. Alternatively, thecompositions may be applied in other dosage forms known to those skilledin the art, such as pre-formed or in situ-formed gels or liposomes.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are may be formulated for surgical implantation on orbeneath the renal capsule. It should be appreciated that liquidcompositions also may be administered by surgical procedures. Inparticular cases, semi-solid or solid pharmaceutical compositions maycomprise semi-permeable gels, lattices, cellular scaffolds and the like,which may be non-biodegradable or biodegradable. For example, in certaincases, it may be desirable or appropriate to sequester the exogenouscells from their surroundings, yet enable the cells to secrete anddeliver biological molecules (e.g., insulin) to surrounding cells or theblood stream. In these cases, cells may be formulated as autonomousimplants comprising living non-native pancreatic β cells or cellpopulation comprising non-native pancreatic β cell surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression.

In other cases, various degradable gels and networks can be used for thepharmaceutical compositions of the present disclosure. For example,degradable materials particularly suitable for sustained releaseformulations include biocompatible polymers, such as poly(lactic acid),poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like.

In other cases, it may be desirable or appropriate to deliver the cellson or in a biodegradable, preferably bioresorbable or bioabsorbable,scaffold or matrix. These typically three-dimensional biomaterialscontain the living cells attached to the scaffold, dispersed within thescaffold, or incorporated in an extracellular matrix entrapped in thescaffold. Once implanted into the target region of the body, theseimplants become integrated with the host tissue, wherein thetransplanted cells gradually become established.

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present disclosure includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven mats,for example, may be formed using fibers comprising a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), foams,and/or poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA)copolymer.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures. In manyof the abovementioned cases, the framework may be molded into a usefulshape. Furthermore, it will be appreciated that non-native pancreatic βcells may be cultured on pre-formed, non-degradable surgical orimplantable devices.

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

In one aspect, the present disclosure provided devices comprising a cellcluster comprising at least one pancreatic β cell. A device providedherein can be configured to produce and release insulin when implantedinto a subject. A device can comprise a cell cluster comprising at leastone pancreatic β cell, e.g., a non-native pancreatic β cell. A cellcluster in the device can exhibit in vitro GSIS. A device can furthercomprise a semipermeable membrane. The semipermeable membrane can beconfigured to retain the cell cluster in the device and permit passageof insulin secreted by the cell cluster. In some cases of the device,the cell cluster can be encapsulated by the semipermeable membrane. Theencapsulation can be performed by any technique available to one skilledin the art. The semipermeable membrane can also be made of any suitablematerial as one skilled in the art would appreciate and verify. Forexample, the semipermeable membrane can be made of polysaccharide orpolycation. In some cases, the semipermeable membrane can be made ofpoly(lactide) (PLA), poly(glycolic acid) (PGA),poly(lactide-co-glycolide) (PLGA), and other polyhydroxyacids,poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyphosphazene, polyamino acids, polyortho esters, polyacetals,polycyanoacrylates, biodegradable polyurethanes, albumin, collagen,fibrin, polyamino acids, prolamines, alginate, agarose, agarose withgelatin, dextran, polyacrylates, ethylene-vinyl acetate polymers andother acyl-substituted cellulose acetates and derivatives thereof,polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinyl imidazole), chlorosulphonated polyolefins, polyethyleneoxide, or any combinations thereof. In some cases, the semipermeablemembrane comprises alginate. In some cases, the cell cluster isencapsulated in a microcapsule that comprises an alginate coresurrounded by the semipermeable membrane. In some cases, the alginatecore is modified, for example, to produce a scaffold comprising analginate core having covalently conjugated oligopeptides with an RGDsequence (arginine, glycine, aspartic acid). In some cases, the alginatecore is modified, for example, to produce a covalently reinforcedmicrocapsule having a chemoenzymatically engineered alginate of enhancedstability. In some cases, the alginate core is modified, for example, toproduce membrane-mimetic films assembled by in-situ polymerization ofacrylate functionalized phospholipids. In some cases, microcapsules arecomposed of enzymatically modified alginates using epimerases, In somecases, microcapsules comprise covalent links between adjacent layers ofthe microcapsule membrane. In some embodiment, the microcapsulecomprises a subsieve-size capsule comprising alginate coupled withphenol moieties. In some cases, the microcapsule comprises a scaffoldcomprising alginate-agarose. In some cases, the SC-β cell is modifiedwith PEG before being encapsulated within alginate. In some cases, theisolated populations of cells, e.g., SC-β cells are encapsulated inphotoreactive liposomes and alginate. It should be appreciated that thealginate employed in the microcapsules can be replaced with othersuitable biomaterials, including, without limitation, polyethyleneglycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronicacid, dextran with ROD, BHD and polyethylene glycol-diacrylate (PEGDA),poly(MPC-co-n-butyl methacrylate-co-4-vinylphenyl boronic acid) (PMBV)and poly(vinyl alcohol) (PVA), agarose, agarose with gelatin, andmultilayer cases of these. In some cases, the device provided hereincomprise extracorporeal segment, e.g., part of the device can be outsidea subject's body when the device is implanted in the subject. Theextracorporeal segment can comprise any functional component of thedevice, with or without the cells or cell cluster provided herein.

VII. Methods of Treating

Further provided herein are methods for treating or preventing a diseasein a subject. A composition comprising the cell clusters or cellsprovided herein or generated according to the methods provided hereincan be administered into a subject to restore a degree of pancreaticfunction in the subject. For example, the cell clusters resemblingendogenous pancreatic islets, or the cells resembling endogenouspancreatic β cells (e.g., non-native pancreatic β cells or SC-β cells)or the precursors thereof can be transplanted to a subject to treatdiabetes.

The methods can comprise transplanting the cell cluster or the celldisclosed in the application to a subject, e.g., a subject in needthereof. The term “transplanting” can refer to the placement of cells orcell clusters, any portion of the cells or cell clusters thereof, or anycompositions comprising cells, cell clusters or any portion thereof,into a subject, by a method or route which results in at least partiallocalization of the introduced cells or cell clusters at a desired site.The cells or cell clusters can be implanted directly to the pancreas, oralternatively be administered by any appropriate route which results indelivery to a desired location in the subject where at least a portionof the implanted cells or cell remain viable. The period of viability ofthe cells or cell clusters after administration to a subject can be asshort as a few hours, e.g. twenty-four hours, to a few days, to as longas several years. In some instances, the cells or cell clusters, or anyportion of the cells or cell clusters thereof, can also betransadministered at a non-pancreatic location, such as in the liver orsubcutaneously, for example, in a capsule (e.g., microcapsule) tomaintain the implanted cells or cell clusters at the implant locationand avoid migration.

As used herein, the term “treating” and “treatment” can refer toadministering to a subject an effective amount of a composition (e.g.,cell clusters or a portion thereof) so that the subject as a reductionin at least one symptom of the disease or an improvement in the disease,for example, beneficial or desired clinical results. For purposes ofthis disclosure, beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptoms, diminishment ofextent of disease, stabilized (e.g., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (e.g., partial or total), whetherdetectable or undetectable. Treating can refer to prolonging survival ascompared to expected survival if not receiving treatment. Thus, one ofskill in the art realizes that a treatment may improve the diseasecondition, but may not be a complete cure for the disease. As usedherein, the term “treatment” includes prophylaxis.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,suhcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

By “treatment,” “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Treatment of Diabetes is determined by standard medical methods. A goalof Diabetes treatment is to bring sugar levels down to as close tonormal as is safely possible. Commonly set goals are 80-120 milligramsper deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. Aparticular physician may set different targets for the patent, dependingon other factors, such as how often the patient has low blood sugarreactions. Useful medical tests include tests on the patient's blood andurine to determine blood sugar level, tests for glycosylated hemoglobinlevel (HbA1c; a measure of average blood glucose levels over the past2-3 months, normal range being 4-6%), tests for cholesterol and fatlevels, and tests for urine protein level. Such tests are standard testsknown to those of skill in the art (see, for example, American DiabetesAssociation, 1998). A successful treatment program can also bedetermined by having fewer patients in the program with complicationsrelating to Diabetes, such as diseases of the eye, kidney disease, ornerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset ofat least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia,diabetic retinopathy, diabetic nephropathy, blindness, memory loss,renal failure, cardiovascular disease (including coronary arterydisease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1week, at least 2 weeks, at least 1 month, at least 2 months, at least 6months, at least 1 year, at least 2 years, at least 5 years, at least 10years, at least 20 years, at least 30 years, at least 40 years or more,and can include the entire lifespan of the subject.

In some aspects, the disclosure relates to a method comprisingimplanting in a subject a device comprising a cell or cell clusterprovided herein (e.g., insulin producing cells), wherein the devicereleases insulin in an amount sufficient for a reduction of bloodglucose levels in the subject. In some embodiments, the insulinproducing cells are glucose responsive insulin producing cells.

In some embodiments, the reduction of blood glucose levels in thesubject, as induced by the transplantation of the cell or cell cluster,or the device provided herein, results in an amount of glucose which islower than the diabetes threshold. In some embodiments, the subject is amammalian subject. In some embodiments, the mammalian subject is human.In some embodiments, the amount of glucose is reduced to lower than thediabetes threshold in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after theimplanting.

As described in detail above, the pharmaceutical compositions of thepresent disclosure can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

A subject that can be treated by the methods herein can be a human or anon-human animal. In some cases, a subject can be a mammal. Examples ofa subject include but are not limited to primates, e.g., a monkey, achimpanzee, a bamboo, or a human. In some cases, a subject is a human. Asubject can be non-primate animals, including, but not limited to, adog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, and thelike. In some cases, a subject receiving the treatment is a subject inneed thereof, e.g., a human in need thereof.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. The terms, “patient” and “subject” are used interchangeablyherein. Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of Type 1diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. Inaddition, the methods described herein can be used to treat domesticatedanimals and/or pets. A subject can be male or female. A subject can beone who has been previously diagnosed with or identified as sufferingfrom or having Diabetes (e.g., Type 1 or Type 2), one or morecomplications related to Diabetes, or a pre-diabetic condition, andoptionally, but need not have already undergone treatment for theDiabetes, the one or more complications related to Diabetes, or thepre-diabetic condition. A subject can also be one who is not sufferingfrom Diabetes or a pre-diabetic condition. A subject can also be one whohas been diagnosed with or identified as suffering from Diabetes, one ormore complications related to Diabetes, or a pre-diabetic condition, butwho show improvements in known Diabetes risk factors as a result ofreceiving one or more treatments for Diabetes, one or more complicationsrelated to Diabetes, or the pre-diabetic condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingDiabetes, one or more complications related to Diabetes, or apre-diabetic condition. For example, a subject can be one who exhibitsone or more risk factors for Diabetes, complications related toDiabetes, or a pre-diabetic condition, or a subject who does not exhibitDiabetes risk factors, or a subject who is asymptomatic for Diabetes,one or more Diabetes-related complications, or a pre-diabetic condition.A subject can also be one who is suffering from or at risk of developingDiabetes or a pre-diabetic condition. A subject can also be one who hasbeen diagnosed with or identified as having one or more complicationsrelated to Diabetes or a pre-diabetic condition as defined herein, oralternatively, a subject can be one who has not been previouslydiagnosed with or identified as having one or more complications relatedto Diabetes or a pre-diabetic condition.

The methods can comprise transplanting the cell cluster to a subjectusing any means in the art. For example the methods can comprisetransplanting the cell cluster via the intraperitoneal space, renalsubcapsule, renal capsule, omentum, subcutaneous space, or viapancreatic bed infusion. For example, transplanting can be subcapsulartransplanting, intramuscular transplanting, or intraportaltransplanting, e.g., intraportal infusion. Immunoprotectiveencapsulation can be implemented to provide immunoprotection to the cellclusters. In some cases, the methods of treatment provided herein cancomprise administer immune response modulator for modulating or reducingtransplant rejection response or other immune response against theimplant (e.g., the cells or the device). Examples of immune responsemodulator that can be used in the methods can include purine synthesisinhibitors like Azathioprine and Mycophenolic acid, pyrimidine synthesisinhibitors like Leflunomide and Teriflunomide, antifolate likeMethotrexate, Tacrolimus, Ciclosporin, Pimecrolimus, Abetimus,Gusperimus, Lenalidomide, Pomalidomide, Thalidomide, PDE4 inhibitor,Apremilast, Anakinra, Sirolimus, Everolimus, Ridaforolimus,Temsirolimus, Umirolimus, Zotarolimus, Anti-thymocyte globulinantibodies, Anti-lymphocyte globulin antibodies, CTLA-4, fragmentthereof, and fusion proteins thereof like Abatacept and Belatacept, TNFinhibitor like Etanercept and Pegsunercept, Aflibercept, Alefacept,Rilonacept, antibodies against complement component 5 like Eculizumab,anti-TNF antibodies like Adalimumab, Afelimomab, Certolizumab pegol,Golimumab, Infliximab, and Nerelimomab, antibodies against Interleukin 5like Mepolizumab, anti-Ig E antibodies like Omalizumab, anti-Interferonantibodies like Faralimomab, anti-IL-6 antibodies like Elsilimomab,antibodies against IL-12 and IL-23 like Lebrikizumab and Ustekinumab,anti-IL-17A antibodies like Secukinumab, anti-CD3 antibodies likeMuromonab-CD3, Otelixizumab, Teplizumab, and Visilizumab, anti-CD4antibodies like Clenoliximab, Keliximab, and Zanolimumab, anti-CD11aantibodies like Efalizumab, anti-CD18 antibodies like Erlizumab,anti-CD20 antibodies like Obinutuzumab, Rituximab, Ocrelizumab andPascolizumab, anti-CD23 antibodies like Gomiliximab and Lumiliximab,anti-CD40 antibodies like Teneliximab and Toralizumab, antibodiesagainst CD62L/L-selectin like Aselizumab, anti-CD80 antibodies likeGaliximab, anti-CD147/Basigin antibodies like Gavilimomab, anti-CD154antibodies like Ruplizumab, anti-BLyS antibodies like Belimumab andBlisibimod, anti-CTLA-4 antibodies like Ipilimumab and Tremelimumab,anti-CAT antibodies like Bertilimumab, Lerdelimumab, and Metelimumab,anti-Integrin antibodies like Natalizumab, antibodies againstInterleukin-6 receptor like Tocilizumab, anti-LFA-1 antibodies likeOdulimomab, antibodies against IL-2 receptor/CD25 like Basiliximab,Daclizumab, and Inolimomab, antibodies against T-lymphocyte (Zolimomabaritox) like Atorolimumab, Cedelizumab, Fontolizumab, Maslimomab,Morolimumab, Pexelizumab, Reslizumab, Rovelizumab, Siplizumab,Talizumab, Telimomab aritox, Vapaliximab, and Vepalimomab.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1—Selection of Cell Lines for SC-β Cell Production

Embryonic stem cell (ESC) cell lines can be used for SC-β cellproduction. Criteria for the ESC cell line selection can includeconsistent expansion in 2D culture system, adaptation to 3D culture,differentiation capacity, and function in vitro and in vivo. Examplecell lines are shown in FIG. 1.

Example 2—2D Culture Criteria for ESC Cell Line

Examples of selected ESC cell line morphology and composition are shownin FIGS. 2A and 2B. In this example, cells expressing non-ESC cellmarker Sox17 and ESC-marker Oct4 were detected by flow cytometry. Morethan 90% of ESC cells were detected in the cell population.

Example 3—Differentiation Protocol of ESC Cell Line

This example shows that exemplary basal culture medium according to thepresent disclosure can have improve differentiation efficiency asqualified by on-target and off-target progenitor populations. As shownin FIG. 3A, basal medium containing an exemplary component can increasepercentage of on-target cells while decrease percentage of off-targetcells. FIG. 3B shows that an exemplary component of the basal culturemedium, human serum albumin (HSA), significantly increased thepercentage of Pdx1-positive cells (Pdx1+) at the end of stage 3, ascompared to differentiating the cells in culture medium containingbovine serum albumin (BSA) instead or culture medium having no serumalbumin (“No SA”), when cells are differentiated following an exemplarydifferentiation protocol according to the present disclosure. At thesame time, the presence of HSA significantly decreased the percentage ofoff-target CDX2-positive (CDX2+) cells at the end of stage 3, ascompared to cells differentiated in culture medium containing BSA or NoSA. The data also demonstrated a concentration-dependent effect of HSAon percentage of Pdx1+ and CDX2+ cells.

Another example as illustrated here demonstrated that an exemplarydifferentiation protocol according to the present disclosure (v10protocol), which comprises differentiating primitive gut tube (PGT)cells into Pdx1-positive pancreatic progenitor cells (Stage 3) byincubating PGT cells in medium containing Activin A (20 ng/mL) and DMH-1(0.25 μM), can significantly increase differentiation efficiency asdetermined by percentage of NKX6.1-positive, C-peptide-positive SC-βcells at the end of Stage 6 differentiation. As shown in FIGS. 4A and4B, percentage of SC-β cells was increased by protocol v10 in comparisonwith other exemplary protocols without the incubation of both Activin Aand DMH-1 at Stage 3 (v8 and v9). 1.5 to 2 fold increase ofNKX6.1-positive, C-peptide-positive SC-β cells SC-β cell percentage wasachieved by using protocol v10 as compared to another exemplaryprotocol. As shown in FIG. 4B, exemplary cell clusters generated by v10protocol comprised more than 35% NKX6.1-positive, C-peptide-positiveSC-β cells.

SC-β cells differentiated using the exemplary methods provided herein(exemplary protocols) resemble the natural pancreatic islet cells. Asshown in image data in FIG. 5, SC-β cells had similar morphology as thenatural islet cells. The images were produced by staining a β cellspecific gene and insulin.

Example 4—Functional Analysis of Differentiated SC-β Cells

Signaling factors (e.g., exemplary differentiation factors disclosedherein, e.g., Activin A, DMH-1, LDN193189, or Alk5i) according topresent disclosure can improve the in vitro function of SC-β cellsgenerated using exemplary methods provided herein.

In one example, flow cytometry analysis showed that the presence orabsence of LDN193189 at Stage 3 changed the cell constituent obtained atStage 4-complete (e.g., at the completion of Stage 4 culture) accordingto one exemplary differentiation method provided herein. As shown inFIG. 6A, in this example, incubation of PGT cells (Stage 2 cells) withLDN193189 (“LDN” in the figure) clearly reduced the percentage ofCDX2-positive cells in the cell population obtained at Stage 4-complete.In the meantime, LDN193189 also promoted chromogranin A (CHGA)expression in Stage 4-complete cells.

In another example, flow cytometry analysis showed that incubation ofthe cells comprising PGT cells (Stage 2 cells) with certain exemplarysignaling factors (e.g., Activin A and DMH-1) affected the cellconstituent obtained at Stage 4-complete (e.g., at the completion ofStage 4 culture) according to one exemplary differentiation methodprovided herein. As shown in FIG. 6B, in this example, incubation of 0.5μM DMH-1 alone reduced the percentage of cells expressing CDX2 butincreased the percentage of cells expressing CHGA as compared to noincubation of DMH-1, similar to the effect of LDN193189 in the otherexample as shown in FIG. 6A. However, co-incubation of 0.5 μM DMH-1 and20 ng/mL Activin A (“AA” in the figure) reduced the percentage ofCDX2-positive cells but also controlled the percentage of CHGA-positivecells under a low level as compared to the Stage 4-complete cellsobtained without any DMH-1 or Activin A.

In another example, incubation of the cells comprising PGT cells (Stage2 cells) with certain exemplary signaling factors (e.g., Activin A andDMH-1) significantly improved the insulin secretion of the cellpopulation obtained at Stage 6 that comprises the SC-β cells. In vitroglucose-stimulated insulin secretion (GSIS) assay was used to quantifythe function of SC-β cells in insulin secretion. As shown in FIG. 6C,addition of the exemplary signaling factors (DMH-1 and Activin A) atStage 3 proved to improve the GSIS reactions in the presence of lowglucose (2.8 mM), high glucose (20 mM), or KCl (30 mM KCl+2.8 mMglucose). As demonstrated by the figure, the exemplary signaling factors(Activin A and DMH-1) significantly increased the GSIS stimulation indexas calculated by insulin secretion in response to high glucosestimulation divided by insulin secretion in response to low glucosestimulation. For instance, the stimulation index of Stage 6 cellsobtained with the exemplary protocol using 20 ng/mL Activin A and 0.25μM DMH-1 at Stage 3 was at least 3 times higher than the Stage 6 cellsobtained with the exemplary protocol that does not use Activin A orDMH-1 at Stage 3. On the other hand, addition of Activin A and DMH-1also significantly increased insulin secretion in response to KClstimulation in a concentration-dependent manner. For example, theexemplary Stage 6 cells obtained with the exemplary protocol using 20ng/mL Activin A and 0.25 μM DMH-1 at Stage 3 secreted at least 3 timeshigher insulin in response to KCl stimulation as compared to the Stage 6cells obtained with the exemplary protocol using no Activin A or DMH-1at Stage 3, while Stage 6 cells obtained with 10 ng/mL Activin A and0.25 μM DMH-1 at Stage 3 secreted about 2 times higher insulin inresponse to the same KCl stimulation.

Example 5—Manufacturing of SC-β Cells in Large Scale

Production of SC-β cells was tested in large-scale cultures, forexample, 0.1 L, 0.5 L, and 3 L cultures as shown in FIGS. 9A-9C. Foldexpansion of cells was increased when the culture size was increased.The population of Oct4 expressing cells was qualified in eachlarge-scale culture, and each of the large-scale cultures produced closeto 100% Oct4 expressing cells.

The produced SC-β cells still maintained the appropriate function (e.g.the ability to sense glucose and secrete insulin) aftercryopreservation. As shown is FIG. 10, fresh SC-β cells andcryopreserved SC-β cells were compared in their ability to sense glucoseand secrete insulin. Cryopreserved SC-β cells exhibited comparableactivity.

Example 6—Treatment of Animal Models with SC-β Cells

SC-β cells were implanted into animal models, optionally by placing in adevice, and implanting the device (e.g. diabetic immunodeficient mice).Tissue samples were obtained after 6 month of transplant. An exampleimage of the tissue sample is shown in FIG. 11. Transplanted SC-β cellgraft contained large numbers of a and β cells. Image was stained usingC-peptide, glucagon, and DAPI.

Encapsulated SC-β cells can cure diabetes in animal models (e.g.diabetic immunodeficient mice). As shown in FIG. 12, mice were firstinduced to have diabetes, encapsulated SC-β cells were then implantedinto mice after inducing diabetes, and finally the SC-β cells wereexplanted after certain time. Blood glucose level was monitored throughthe whole process. After inducing diabetes, the blood glucose level wentup to a level much higher than diabetes threshold. After implantation ofSC-β cells, blood glucose level was dropped to below the diabetesthreshold level. After explanation, the blood glucose level increasedback to the high level.

In another example, mice were implanted with a device filled with anexemplary SC-β cell population that was developed in vitro. FIG. 14shows an image of the in vitro developed SC islets before encapsulationof the cells in the capsule device, as well as an image of the cellsthat were removed from the device 3 months after implantation into amouse. As demonstrated by the C-peptide and Glucagon immunostaining,these encapsulated SC-islets show high number of viable β cellspost-implant in mice.

Example 7—Constituent Analysis of Differentiated SC-β Cells

Exemplary differentiated SC-β cell populations were examined by flowcytometry to analyze their constituents. According to some aspect of thepresent disclosure, three differentiated populations were generated bythree different protocols, and their flow cytometry analysis results areshown in FIG. 13. Cell population 1 was generated using conventionalprotocol, while cell populations 2 and 3 were generated using exemplaryprotocols as provided herein. As shown in FIG. 13, cell populations 1,2, and 3 are all mixtures of different cell types, as suggested bydifferent marker expression patterns. Notably, cell population 3 had thehighest β cell population (NKX6.1⁺/C-peptide⁺).

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1-103. (canceled)
 104. An in vitro method comprising contacting apopulation of Pdx1-negative primitive gut tube cells with a compositioncomprising a BMP signaling pathway inhibitor, wherein the BMP signalingpathway inhibitor is a compound of Formula A,

or a derivative, analogue, or variant thereof.
 105. The method of claim104, wherein the composition further comprises a growth factor from thetransformation growth factor β (TGF-β) superfamily.
 106. The method ofclaim 105, further comprising differentiating the population ofprimitive gut tube cells to generate a cell cluster comprisingnon-native pancreatic β cells capable of having a glucose stimulatedinsulin secretion (GSIS) response.
 107. The method of claim 106, whereinthe differentiating of the population of primitive gut tube cellsgenerates a cell cluster that comprises a higher percentage ofnon-native pancreatic β cells capable of having a GSIS response ascompared to a cell cluster differentiated from a comparable populationof primitive gut tube cells without the contacting with the compositioncomprising the BMP signaling pathway inhibitor and the growth factorfrom the transformation growth factor β (TGF-β) superfamily.
 108. Themethod of claim 106, wherein the differentiating of the population ofprimitive gut tube cells generates a cell cluster that comprisesnon-native pancreatic β cells that has a higher glucose-stimulatedinsulin secretion (GSIS) stimulation index than a cell clustercomprising non-native pancreatic β cells differentiated from acomparable population of primitive gut tube cells without the BMPsignaling pathway inhibitor or the growth factor from the transformationgrowth factor β (TGF-β) superfamily.
 109. The method of claim 108,wherein the GSIS stimulation index is calculated as a ratio of insulinsecretion in response to a first glucose concentration to insulinsecretion in response to a second glucose concentration, and whereinsaid first glucose concentration is about 10 to about 50 mM, and saidsecond glucose concentration is about 1 mM to 5 mM.
 110. The method ofclaim 109, wherein the GSIS stimulation index of the cell clustercomprising non-native pancreatic β cells is at least about 3 fold higherthan that of the cell cluster comprising non-native pancreatic β cellsdifferentiated from the comparable population of primitive gut tubecells.
 111. The method of claim 104, further comprising differentiatingthe Pdx-1-positive pancreatic progenitor cells into a cell clustercomprising NKX6.1-positive pancreatic progenitor cells.
 112. The methodof claim 111, wherein the cell cluster comprises at least 50%Pdx-1-positive, NKX6.1-positive cells as measured by flow cytometry.113. The method of claim 111, wherein the cell cluster comprisingNKX6.1-positive pancreatic progenitor cells comprises at most 30%CHGA-positive cells as measured by flow cytometry.
 114. The method ofclaim 111, wherein the cell cluster comprising NKX6.1-positivepancreatic progenitor cells comprises at most 30% CDX2-positive cells asmeasured by flow cytometry.
 115. The method of claim 111, wherein thecell cluster comprising NKX6.1-positive pancreatic progenitor cellscomprises at least 50% PDX1-positive, NKX6.1-positive pancreaticprogenitor cells, at most 25% CDX2-positive, NKX6.1-positive cells, andat most 10% CHGA-positive cells, as measured by flow cytometry.
 116. Themethod of claim 105, wherein the growth factor from the transformationgrowth factor β (TGF-β) superfamily is selected from the groupconsisting of an Inhibin, an Activin, a Mullerian inhibiting substance(MIS), a bone morphogenic protein (BMP), decapentaplegic (dpp), Vg-1,monoclonal nonspecific suppressor factor (MNSF), growth differentiatingfactor 8 (GDF8), and growth differentiating factor 11 (GDF11).
 117. Themethod of claim 105, wherein the growth factor from the transformationgrowth factor β (TGF-β) superfamily comprises Activin A.
 118. The methodof claim 117, wherein the composition comprises about 2 ng/mL to about50 ng/mL of Activin A.
 119. The method of claim 104, wherein thecomposition comprises about 0.1 μM to about 0.3 μM of the BMP signalinginhibitor.
 120. The method of claim 105, further comprising contactingthe population of primitive gut tube cells with a growth factor fromfibroblast growth factor (FGF) family.
 121. The method of claim 105,further comprising contacting the population of primitive gut tube cellswith a Sonic Hedgehog (SHH) pathway inhibitor.
 122. The method of claim105, further comprising contacting the population of primitive gut tubecells with a Retinoic Acid (RA) signaling pathway activator.
 123. Themethod of claim 105, further comprising contacting the population ofprimitive gut tube cells with a protein kinase C activator.
 124. Themethod of claim 105, further comprising contacting the population ofprimitive gut tube cells with a Rho kinase (ROCK) inhibitor.
 125. Themethod of claim 105, further comprising contacting the primitive guttube cells with a growth factor from fibroblast growth factor (FGF)family, a Sonic Hedgehog (SHH) pathway inhibitor, a Retinoic Acid (RA)signaling pathway activator, a protein kinase C activator, and a ROCKinhibitor.
 126. The method of claim 105, further comprising contactingthe primitive gut tube cells with keratinocyte growth factor (KGF),SANT1, retinoic acid, PdbU, and thiazovivin.
 127. A compositioncomprising a population of Pdx1-negative primitive gut tube cells and aBMP signaling pathway inhibitor, wherein the BMP signaling pathway is acompound of Formula (A),

or a derivative, analogue, or variant thereof.
 128. An in vitrocomposition comprising a population of pancreatic progenitor cells;wherein at least 50% of the cells in the population are Pdx1-positiveand NKX6.1-positive; wherein at most 30% of the cells in the populationare CHGA-positive; and wherein at most 15% of the cells in thepopulation are CDX2-positive as measured by flow cytometry.
 129. Adevice comprising an in vitro cell cluster, wherein at least 40% of thecells in the cell cluster are NKX6.1-positive and C-peptide-positive.130. A method of treating a subject in need thereof, wherein the methodcomprises administering to the subject the device of claim 129.